U.S. patent application number 16/966204 was filed with the patent office on 2022-06-09 for methods of treating optic nerve diseases using neural progenitor cell growth factors.
This patent application is currently assigned to University of Maryland, Baltimore. The applicant listed for this patent is Steven BERNSTEIN, Candace L. KERR, REGENERATIVE RESEARCH FOUNDATION, Sally A. TEMPLE, University of Maryland, Baltimore. Invention is credited to Steven Lance BERNSTEIN, Candace L. KERR, Sally TEMPLE STERN.
Application Number | 20220175844 16/966204 |
Document ID | / |
Family ID | 1000006198052 |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220175844 |
Kind Code |
A1 |
BERNSTEIN; Steven Lance ; et
al. |
June 9, 2022 |
METHODS OF TREATING OPTIC NERVE DISEASES USING NEURAL PROGENITOR
CELL GROWTH FACTORS
Abstract
Optic nerve lamina region neural progenitor cells (ONLR-NPCs)
are provided. Such cells secrete growth factors and survival
factors that can be used in the treatment of optic nerve diseases,
such as glaucoma. Also provided are methods of treating or
preventing optic nerve diseases, such as glaucoma, using one or
more factors secreted by ONLR-NPCs, combinations of factors
secreted by ONLR-NPCs, or culture media conditioned by
ONLR-NPCs.
Inventors: |
BERNSTEIN; Steven Lance;
(Chevy Chase, MD) ; KERR; Candace L.;
(Westminster, MD) ; TEMPLE STERN; Sally;
(Slingerlands, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BERNSTEIN; Steven
KERR; Candace L.
TEMPLE; Sally A.
University of Maryland, Baltimore
REGENERATIVE RESEARCH FOUNDATION |
Chevy Chase
Westminster
Slingerlands
Baltimore
Rensselaer |
MD
MD
NY
MD
NY |
US
US
US
US
US |
|
|
Assignee: |
University of Maryland,
Baltimore
Baltimore
MD
REGENERATIVE RESEARCH FOUNDATION
Rensselaer
NY
|
Family ID: |
1000006198052 |
Appl. No.: |
16/966204 |
Filed: |
February 1, 2019 |
PCT Filed: |
February 1, 2019 |
PCT NO: |
PCT/US19/16303 |
371 Date: |
July 30, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62625593 |
Feb 2, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1825 20130101;
A61K 38/1841 20130101; A61K 38/185 20130101; A61K 38/18 20130101;
A61K 35/30 20130101; A61K 38/1866 20130101; A61P 27/06
20180101 |
International
Class: |
A61K 35/30 20060101
A61K035/30; A61K 38/18 20060101 A61K038/18; A61P 27/06 20060101
A61P027/06 |
Goverment Interests
STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0001] This invention was made with government support under Grant
Number EY015304 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of treating or preventing an optic nerve disease in a
subject comprising administering to a subject in need thereof a
therapeutically effective amount of a composition comprising one or
more of: (i) a population of optic nerve lamina region-neural
progenitor cells (ONLR-NPCs), (ii) ONLR-NPC conditioned media,
(iii) an ONLR-NPC lysate, (iv) an ONLR-NPC extract, and (v) one or
more factors secreted by ONLR-NPCs, thereby treating or preventing
an optic nerve disease in the subject.
2. The method of claim 1, wherein the optic nerve disease is
selected from the group consisting of open-angle glaucoma,
angle-closure glaucoma, optic nerve hypoplasia, optic nerve
hypomyelination, regional axonal dysfunction, nonarteritic anterior
ischemic optic neuropathy (NAION), and optic neuritis.
3. The method of claim 1, wherein the composition is administered
to the eye of the subject.
4. The method of claim 1, wherein the composition is administered
to the subject via one or more of topical application,
subconjunctival injection, intravitreal injection, and retrobulbar
injection.
5. The method of claim 1, wherein the composition administered to
the subject is a pharmaceutical formulation comprising a
pharmaceutically acceptable carrier.
6. The method of claim 1, wherein the subject is a human.
7. The method of claim 1, wherein the ONLR-NPCs are nestin(+),
SOX2(+), GFAP(+), NG2(-) cells.
8. The method of claim 1, wherein the ONLR-NPCs are nestin(+),
SOX2(+), GFAP(+), NG2(-), SOX1(+), vimentin(+), BDNF(+) cells.
9. The method of claim 1, wherein the ONLR-NPCs express one or more
of Latent Transforming Growth Factor-Beta 1 (TGF-.beta.1),
Connective Tissue Growth Factor (CTGF), Nerve Growth Factor (NGF),
Fibroblast Growth Factor (FGF-1), Vascular Endothelial Growth
Factor (VEGF), Mesenchymal Astrocyte Neurotrophic Factor (MANF) and
Insulin-like Growth Factors-1 and -2 (IGF-1 and IGF-2).
10. The method of claim 1, wherein the ONLR-NPCs express at least
five growth factors selected from the group consisting of VGF,
TGF.beta.1, LTBP1, LTBP2, PDGFB, CTGF, FGF11, NDNF, PDGFC,
TGF.beta.2, NGF, FGF1, Midkine, VEGFA, MANF, IGF-1, and IGF-2.
11. The method of claim 1, wherein the ONLR-NPCs express of VGF,
TGF.beta.1, LTBP1, LTBP2, PDGFB, CTGF, FGF11, NDNF, PDGFC,
TGF.beta.2, NGF, FGF1, Midkine, VEGFA, MANF, IGF-1, and IGF-2.
12. The method of claim 1, wherein the ONLR-NPCs express of one or
more of the proteins listed in Table 2.
13. The method of claim 1, wherein the ONLR-NPCs express each of
the proteins listed in Table 2.
14. The method of claim 1, wherein the composition comprising one
or more factors secreted by ONLR-NPCs comprises one or more of
Latent Transforming Growth Factor-Beta 1 (TGF-.beta.1), Connective
Tissue Growth Factor (CTGF), Nerve Growth Factor (NGF), Fibroblast
Growth Factor (FGF-1), Vascular Endothelial Growth Factor (VEGF),
Mesenchymal Astrocyte Neurotrophic Factor (MANF) and Insulin-like
Growth Factors-1 and -2 (IGF-1 and IGF-2).
15. The method of claim 1, wherein the composition comprising one
or more factors secreted by ONLR-NPCs comprises each of Latent
Transforming Growth Factor-Beta 1 (TGF-.beta.1), Connective Tissue
Growth Factor (CTGF), Nerve Growth Factor (NGF), Fibroblast Growth
Factor (FGF-1), Vascular Endothelial Growth Factor (VEGF),
Mesenchymal Astrocyte Neurotrophic Factor (MANF) and Insulin-like
Growth Factors-1 and -2 (IGF-1 and IGF-2).
16. The method of claim 1, wherein the composition comprising one
or more factors secreted by ONLR-NPCs comprises at least five of
growth factors selected from the group consisting of VGF,
TGF.beta.1, LTBP1, LTBP2, PDGFB, CTGF, FGF11, NDNF, PDGFC,
TGF.beta.2, NGF, FGF1, Midkine, VEGFA, MANF, IGF-1, and IGF-2.
17. The method of claim 1, wherein the composition comprising one
or more factors secreted by ONLR-NPCs comprises each of VGF,
TGF.beta.1, LTBP1, LTBP2, PDGFB, CTGF, FGF11, NDNF, PDGFC,
TGF.beta.2, NGF, FGF1, Midkine, VEGFA, MANF, IGF-1, and IGF-2.
Description
SEQUENCE LISTING
[0002] A sequence listing in electronic (ASCII text file) format is
filed with this application and incorporated herein by reference.
The name of the ASCII text file is "2020_2122A_ST25.txt"; the file
was created on Dec. 2, 2020; the size of the file is 7 KB.
BACKGROUND
[0003] The primary cause of blindness in humans is glaucoma, an
optic neuropathy characterized by progressive degeneration of the
optic nerve due to increased intraocular pressure over time.
Glaucoma is a progressive disease which can lead to permanent
vision loss within a few years in the absence of treatment. There
are two types of glaucoma, open-angle glaucoma and angle-closure
glaucoma. The majority of cases in the United States and Western
Europe are the former type, while the latter is most common in
China and other Asian countries. Both types of glaucoma result from
improper drainage of aqueous humor from the eye (Weinreb et al.,
2016). While angle-closure glaucoma results from improper drainage
of aqueous humor from the eye (Weinreb et al., 2016) the causes of
open-angle glaucoma are now known to be more complex than simple
increase in intraocular pressure (Levin, 2005).
[0004] The pathophysiology of glaucoma is not well understood, but
intraocular pressure is thought to contribute to damage to the
optic nerve head with a corresponding loss of retinal ganglion
cells. Other causes of damage in open-angle glaucoma include loss
of neurotrophic factors and changes in underlying supporting cells
such as glia (Almasieh et al., 2012). Current treatments for
glaucoma focus only on intraocular pressure reduction and this
approach is only partly successful. Alternative and/or supplemental
treatments for glaucoma and related optic nerve diseases are
needed. The present disclosure is directed to such needs and other
important goals.
BRIEF SUMMARY
[0005] The optic nerve (ON) is a paired nerve that transmits visual
information from the retina of each eye to the brain. The ON is
composed of retinal ganglion cell axons and glial cells, with
around one million axons of retinal ganglion cells present in the
retina of each human eye. Axons passing through the ON that extend
from the eye to brain are myelinated in the anterior ON below the
optic nerve lamina region (ONLR).
[0006] The ONLR is a complex, specialized, unmyelinated boundary
tissue present in primates and most mammals that contains a
vascular plexus with retinal, choroidal and optic nerve
contributions. The ONLR is located at the junction of the optic
nerve and the eye, partially embedded in the eye wall (FIGS. 1A
& 1B), and it can be further characterized as the region in
which unmyelinated retinal ganglion cell axons are myelinated as
they combine to form the optic nerve (ON). Animals without an ONLR,
such as rabbits, have myelinated retinas suggesting that the ONLR
acts as a barrier to the myelination process.
[0007] The multiple functions of the ONLR include blocking
intraocular myelination, enabling myelination of the growing axons
in postnatal individuals, and modulating fluid pressure differences
between eye and brain. The ONLR is also hypothesized to be the
presumptive primary lesion in age-related open-angle glaucoma
(OAG). The precise mechanisms enabling the ONLR to perform all of
these widely disparate functions are unknown.
[0008] As reported herein, it has been discovered that a
mitotically active, age-depletable neural progenitor cell (NPC)
population is present in the ONLR of humans and rodents. ONLR-NPCs
have a unique genetic signature and they produce growth and
survival factors that play a key role in maintaining ocular health
and function. It appears that ONLR-NPCs are lost during aging, in
glaucoma, and in other optic nerve diseases. Loss of the ONLR-NPCs
and therefore loss of the growth and survival factors secreted from
these cells likely contributes to optic nerve diseases that become
increasingly severe over time. Replacement of these secreted
factors in the eye, either alone or in combination with other
treatments (e.g., those that reduce ocular pressure), may serve to
improve the treatment of glaucoma and other optic nerve
diseases.
[0009] Using reporter mice with conditional gene expression or
inducible selective knockouts, it is shown herein that ONLR-NPCs
can generate all macroglial cell forms in the anterior ON. Using a
green fluorescent protein reporter, it is shown herein that
ONLR-NPCs provide the myelinating oligodendrocytes as growing axons
emerge from the eye. The ONLR acts as a gliogenic processing
center, ultimately supplying the growing ON with glial cells as
growing unmyelinated axons emerge from the eye. Early ONLR-NPC loss
results in ON hypoplasia, regional axonal dysfunction and
hypomyelination. In mature animals, ONLR-NPCs may also enable glial
replacement and remyelination. Age-related ONLR-NPC depletion may
help explain why focal ON diseases such as primary open-angle
glaucoma progress in severity during aging.
[0010] ONLR-NPCs, as defined herein, form the basis of the present
invention. The invention thus includes, but is not limited to,
ONLR-NPCs, cultures of ONLR-NPCs, ONLR-NPC conditioned media,
ONLR-NPC lysates, ONLR-NPC extracts, and compositions comprising
one or more of the noted cells, cultures, media, lysates and
extracts.
[0011] The invention also includes methods of treating or
preventing optic nerve diseases, such as glaucoma, using one or
more of ONLR-NPCs, cultures of ONLR-NPCs, ONLR-NPC lysates,
ONLR-NPC extracts, and compositions comprising one or more of the
noted cells, cultures, lysates and extracts.
[0012] ONLR-NPCs secrete particular factors, such as growth factors
and survival factors, and the invention is directed to the use of
these secreted factors as well. The invention thus includes methods
of treating or preventing optic nerve diseases, such as glaucoma,
using one or more factors secreted by ONLR-NPCs, combinations of
factors secreted by ONLR-NPCs, or culture media conditioned by
ONLR-NPCs. The factors secreted by ONLR-NPCs include, but are not
limited to, growth factors and survival factors.
[0013] In particular, and in a first embodiment, the invention is
directed to optic nerve lamina region neural progenitor cells
(ONLR-NPCs). The ONLR-NPCs of this and the other embodiments and
aspects of the invention are characterized, for example, as
nestin(+), SOX2(+), GFAP(+), NG2(-) cells. The invention includes
single ONLR-NPCs as well as populations of ONLR-NPCs. In one
aspect, the invention is directed to an in vitro cell culture
comprising one or more ONLR-NPCs. In another aspect, the invention
is directed to an in vitro cell culture consisting of one or more
ONLR-NPCs.
[0014] In a second embodiment, the invention is directed to
ONLR-NPC conditioned media, obtained from a culture of ONLR-NPCs,
such as an in vitro cell culture of ONLR-NPCs. The ONLR-NPC
conditioned media may be prepared by growing the ONLR-NPCs for
several days in a defined tissue culture medium containing
Fibroblast Growth Factors-2 and -4. The ONLR-NPC conditioned media
may be collected by decanting the sterile medium, followed by
concentration and dialysis or gel filtration to eliminate both
uninvolved medium, ions and large molecular weight compounds such
as bovine serum albumen.
[0015] In a third embodiment, the invention is directed to an
ONLR-NPC lysate. ONLR-NPC lysates comprise the material collected
from ONLR-NPCs that are broken apart by mechanical, chemical means
or physical means. ONLR-NPC lysates may be prepared by collecting
the ONLR-NPCs grown in appropriate medium by centrifugation and
washing these cells, followed by lysis via mechanical, chemical
means or physical means.
[0016] In a fourth embodiment, the invention is directed to an
ONLR-NPC extract. ONLR-NPC extracts comprise some or all of the
growth factors generated and/or secreted by the ONLR-NPCs. The
ONLR-NPC extracts may be a collection of growth factors isolated
from cells, or a collection of selected growth factors known to be
produced by ONLR-NPCs but produce by means other than the ONLR-NPCs
and combined to form an "artificial" ONLR-NPC extract. ONLR-NPC
extracts may thus be prepared by direct isolation of growth factors
from the ONLR-NPCs via lysis and recovery or via collection from
the conditioned medium, as well as by combining selected individual
growth factors into an "artificial" ONLR-NPC extract.
[0017] In a fifth embodiment, the invention is directed to methods
of treating or preventing an optic nerve disease. In one aspect the
method comprises administering to a subject in need thereof a
therapeutically effective amount of a composition comprising one or
more of:
[0018] (i) a population of ONLR-NPCs,
[0019] (ii) ONLR-NPC conditioned media,
[0020] (iii) an ONLR-NPC lysate,
[0021] (iv) an ONLR-NPC extract, and
[0022] (v) one or more factors secreted by ONLR-NPCs,
thereby treating or preventing an optic nerve disease in the
subject.
[0023] In one aspect, the optic nerve disease is open-angle
glaucoma. In another aspect, the optic nerve disease is
angle-closure glaucoma. In a further aspect, the optic nerve
disease is optic nerve hypoplasia, optic nerve hypomyelination,
regional axonal dysfunction, nonarteritic anterior ischemic optic
neuropathy (NAION), or optic neuritis. In one aspect, the
administering is administration to the eye of the subject via means
that include, but are not limited to, topical application (e.g. eye
drops), subconjunctival injection, intravitreal injection, and
retrobulbar injection.
[0024] As indicated above, the ONLR-NPCs of each embodiment and
aspect of the invention are characterized based on the expression
or lack of expression of certain markers by the cells. For example,
in some aspects and embodiments, the ONLR-NPCs are characterized as
nestin(+), SOX2(+), GFAP(+), NG2(-) cells. In a related aspect, the
ONLR-NPCs are characterized as nestin(+), SOX2(+), GFAP(+), NG2(-),
SOX1(+), vimentin(+), BDNF(+) cells.
[0025] In some aspects, the ONLR-NPCs are further characterized
based on their ability to secrete one or more growth factors. These
growth factors include Latent Transforming Growth Factor-Beta 1
(TGF-.beta.1), Connective Tissue Growth Factor (CTGF), Nerve Growth
Factor (NGF), Fibroblast Growth Factor (FGF-1), Vascular
Endothelial Growth Factor (VEGF), Mesenchymal Astrocyte
Neurotrophic Factor (MANF) and Insulin-like Growth Factors-1 and -2
(IGF-1 and IGF-2). In a related aspect, the ONLR-NPCs secrete at
least five of the growth factors listed in Table 1 (i.e., VGF,
TGF.beta.1, LTBP1, LTBP2, PDGFB, CTGF, FGF11, NDNF, PDGFC,
TGF.beta.2, NGF, FGF1, Midkine, VEGFA, MANF, IGF-1, and IGF-2).
Alternatively or in addition, the ONLR-NPCs express at least five
of the additional proteins listed in Table 2. In a further related
aspect, the ONLR-NPCs secrete each of the growth factors listed in
Table 1, or express each of the proteins listed in Table 2, or each
of the growth factors and proteins listed in Tables 1 and 2.
[0026] In one aspect, the composition comprises one or more factors
secreted by ONLR-NPCs, wherein the factors are selected from the
group consisting of Nerve Growth Factor (NGF), Latent Transforming
Growth Factor-Beta 1 (TGF-.beta.1), Fibroblast Growth Factor 1
(FGF1), Vascular Endothelial Growth Factor (VEGF), Mesenchymal
Astrocyte Neurotrophic Factor (MANF), Connective Tissue Growth
Factor (CTGF), and Insulin-like Growth Factors-1 and -2 (IGF-1 and
IGF-2). In a related aspect, the composition comprises each of
these factors. In a further related aspect, the composition
comprises one or more factors secreted by ONLR-NPCs, wherein the
factors are selected from the group set forth in Table 1. In a
related aspect, the composition comprises each of these
factors.
[0027] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described herein, which form the subject of the claims of
the invention. It should be appreciated by those skilled in the art
that any conception and specific embodiment disclosed herein may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that any description, figure, example, etc. is
provided for the purpose of illustration and description only and
is by no means intended to define the limits the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0028] FIG. 1. ONLR Characterization in vivo. A. Schematic of human
ONLR vascularization. The ONLR receives inputs from choroid, ON
pial vessels, and retinal vessels in the vicinity of the nerve. B.
H&E longitudinal section of the mouse ONLR. Retinal neurons
define the outer border of the ONLR. C. Two-photon micrograph of
fluorescein-filled rodent ONLR vessels. ONLR vasculature is more
complex than the overlying retina or ON. Total capillary area
quantification of the three areas is shown in the inset. Retina (R)
and ONLR (L) have equivalent capillary areas, compared with distal
optic nerve (N). Graph constructed from data in (Balaratnasingam et
al., 2014). D. Nestin (red)/myelin (O.sub.4 antibody; green)
localization define the ONLR. Nestin expression is seen as a
gradient that declines anterograde, while myelination below the
ONLR reveals a countergradient. E. AQP4 (in green)/nestin (in red)
confocal colocalization shows reduced ONLR-AQP4, compared with
retina or distal ON. F. ONLR-nestin/GFAP co-localization in the
ONLR. Red: Nestin. Green: GFAP. Scale bar: 20 um. G. SOX2(+) nuclei
are qualitatively increased in the nestin-enriched ONLR
(arrowheads), and SOX2(+) nuclei are present in reduced numbers in
the distal ON (FIG. 1G inset: ON). H. Quantification of SOX2(+)
nuclei in ONLR and ON. The ONLR is enriched in SOX2 nuclei, in
terms of both area (left bars), and SOX2-nuclear ratio (right
bars). I. Ki67-mitotic immunoanalysis in the ONLR and ON. There is
a qualitative increase in the number of mitotic cells in the 60 d
mouse ONLR (arrows), compared with the ON. Inset: Ki67(+) nuclei
quantification in 60 d ONLR and ON. ONLR possesses more mitoses,
and more mitoses per unit area, than in distal ON. J. ONLR
SOX2/GFAP localization. GFAP expression is stronger in ONLR than
the ON, and GFAP cytoplasmic signal is present in cells surrounding
SOX2(+) nuclei, suggesting that the ONLR SOX2(+) cells express both
proteins. Distal ON (inset) reveals that cells with SOX2(+) nuclei
lack GFAP expression. K. NG2 expression is present in the anterior
ON adjacent to the ONLR, while the ONLR is deficient in NG2
expression (lines delineate the NG2 band). NG2 expression is also
diffusely present throughout the distal ON. Inset: Unmyelinated
ONLR defined with nestin (in red) and ON by myelin (O.sub.4: in
green) L. Higher magnification reveals that NG2(+) cells in the ON
have extended cytoplasmic fibrils (arrows), consistent with OPCs.
Scale bars in FIG. 1C, 1F, 1L: 20 um. FIG. 1E, 1G, 1J: 50 um.
[0029] FIG. 2. Characterization of murine ONLR cell cultures and
differential gene expression of ONLR. A. 21 d ONLR culture (no
supplementation). Cells exhibit poor growth, and rapid
differentiation into GFAP(+) astrocytes and O.sub.4(+)
oligodendrocytes (inset). B. 21 d culture of ONLR cells, matrigel
supplementation. Cells generated colonies; cells had extended
processes consistent with neural and/or astrocytic morphology.
Inset: Attached neurosphere on a coated plate. C. Early ONLR
neurospheres. Inset shows subventricular zone (SVZ)-derived
neurospheres from the same animals for comparison. Scale: 100 um.
D. ONLR cells grown on matrigel express NPC markers nestin (green)
and SOX2 (red). Left inset: individual ONLR-NPCs express both
markers. Right inset: nestin(+)/SOX2(+) SVZ-NPCs (arrows) from the
same animal grown in similar conditions. E. Neurosphere formation
quantification from pup (17 d) and adult (60 d) ONLR- and distal ON
segments. Neurosphere formation ability is greatest in ONLR of both
ages. F. Cultured ONLR-NPCs express SOX2 and GFAP (arrows). GFAP
(in red), SOX2 (in green). G. SOX2/A2B5 co-expression. H.
Nestin/NG2 expression. There is minimal colocalization and cells
strongly expressing NG2 do not express nestin (arrowhead),
suggesting that nestin(+) progenitors give rise to OPCs. I.
SOX2/NG2 expression in one-month cell cultures. NG2(+) (green)
cells have multiple cytoplasmic projections, and SOX2(+) nuclei (in
red). J. Nonprogenitor A2B5(+) cells (red) alongside NPC-derived
OPCs. These cells have a flattened, monotonous morphology. OPCs are
A2B5(+)/NG2(+). K. Tuj1 (early neuronal) expression in 21 d ONLR
cultures (arrow). L. ONLR cultures after differentiation with FBS.
Nestin expression is absent. Cells rapidly develop into either
O.sub.4(+) (red) myelinating cells with prolific membranes
consistent with oligodendrocytes, or GFAP(+) (green) cells with
astrocyte-consistent morphology. M. Expression characteristics
table of the two main cell types found in non-differentiated ONLR
cultures (NPCs: 10% of cells; non-NPCs: 90% of cells). ONLR-NPCs
simultaneously express nestin, SOX2 and GFAP, while non-NPCs do
not. N. rqPCR of total RNA obtained from retina, ON, and ONLR. ONLR
expresses the highest level of nestin, and expresses other
progenitor-expressed proteins such as Sox1 and Sox2.
[0030] FIG. 3. GFP induction patterns in SOX2-Cre reporter mice. A.
Schematic of 4OHT/Tamoxifen induction in 19 d PN SOX2-GFP animals.
The unrecombined ROSA26 locus constitutively expresses td-Tomato
(red cytoplasm; background cells in panel 3B). Tamoxifen enables
Cre recombinase expression in SOX2-expressing cells, with
recombined cells expressing GFP in parents and their progeny.
Animals were injected systemically (IP) with tamoxifen or locally
(retrobulbar) with 4OHT (Schematic, FIG. 3A; orange arrow). Tissues
were collected either at 14 d post-induction (33 d postnatal brain;
FIG. 3B and ONLR; FIG. 3C), or 30 d (53 d postnatal; FIG. 3D). B.
Hippocampal GFP expression 14 d post-induction. GFP(+) cells are
present on td-Tomato background. C. ONLR-SOX2-driven GFP expression
14 d post-induction. GFP expression is low, and limited to the
ONLR. D. ONLR SOX2-driven GFP expression 30 d post-induction.
Inset: GFP is present in short processes parallel to the long axis
of the ON, corresponding to myelin segments (arrowheads). E.
SOX2-GFP expression is clustered in ONLR-nestin(+) cells (arrows).
GFP expression is present in ONLR and in the anterior ON. inset:
GFP expression in myelin-containing structures in the ON parallel
to the axis. F. 30 um Z-stack ONLR cross-section in a SOX2-GFP
animal 30 d post-induction. ONLR-GFP expression is concentrated in
large cells with a stellate morphology. Scale bar in G: 50 um; all
other photos: 100 um.
[0031] FIG. 4. Late postnatal deletion of SOX2(+)/Nestin(+)
ONLR-NPCs affects postnatal ON growth and myelination. A.
Transgenic cross and induction schedule. SOX2-DTA animals treated
19 d PN with either vehicle or 4OHT, and analyzed 14 d
post-treatment (33 d). B and C. Gross ON appearance (Vehicle: V and
Experimental: 4OHT) 14 d post-treatment. 4OHT-treated ONs are
thinner (arrowheads), and the contralateral post-chiasmal optic
tract, (axons of the treated nerve) is reduced (arrow), compared
with vehicle treated ONs. D-G: Immunohisto-chemical analysis: Loss
of SOX2 and nestin expression in the ONLR of 4OHT-induced SOX2-DTA
nerves. D and E: Nestin expression. Reduced nestin immunoreactivity
in 4OHT treated nerves (E); compare with vehicle, D. Low power
magnification (Insets in D and E) indicate the relative level of
nestin expression in the anterior ONs. F and G: SOX2 expression.
Vehicle treated nerve (F): left panel; 4OHT treated nerve (G) right
panel. Reduced SOX2(+) nuclear staining in the 4OHT treated nerve.
Inset, G: SOX2(+) quantification in the ONLR of WT (white bar) vs
4OHT treated nerves (Black bar); results.+-.sem; n=3 animals/group.
H. ON ultrastructural analysis-strategy schematic after vehicle or
4OHT-DTA induction in SOX2-DTA double mutants. Letters indicate the
analyzed ON regions. I-L: TEM ultrastructure 14 d post-induction.
I. Anterior ON, 4OHT treated (1.5 mm from the eye). J. Distal ON,
4OHT treated (5 mm from the eye). L: Anterior ON, vehicle treated.
K. Distal ON, vehicle treated. Axons in vehicle treated anterior
and distal ON regions are closely packed, with dense myelination.
Axons in the anterior 4OHT treated ONs have reduced packing
density, and increased extracellular vesicles, compared to vehicle
ONs and more distal portion of the same nerve. The axon anterior
segments have reduced myelination, compared to more distal
segments. Scale bar in I: 500 nm. M. Mean axon diameter
quantification of anterior and posterior ON segments from Vehicle
(white bars) and 4OHT treated (black bars) nerves. .+-.sd. N. Mean
G-ratios of vehicle (white bars) and 4OHT treated (black bars).
.+-.sem; n=3 animals. A statistically significant increase in the
G-ratio of 4OHT treated nerves occur in the anterior ON in animals
treated at 19 d postnatally, after local injection of 5 ul of
vehicle (V) or 4OHT. Data from 6 random 164 um.sup.2 fields from
each nerve region. O. Individual G-ratios for the different axon
size classes from vehicle (white bars) and 4-OHT treated (black
bars) nerves. There was an increase in the G-ratio for all size
classes of 4-OHT treated ONs, and a loss of the smallest axon
diameter class in the anterior segment of 4-OHT treated nerves
(absence of black bar). +/-sd.
[0032] FIG. 5. Age-related NPC depletion in mouse and human ONLR,
and neurosphere development from human donors. A. SOX2 nuclear
expression in 7 mo mouse ONLR. SOX2(+) nuclei are present in nests
within the ONLR, and in high numbers in the anterior ON. B. SOX2
nuclear expression in a 2 y (24 mo) mouse ONLR. There are fewer
SOX2(+) nuclei in both ONLR and anterior ON, compared with 7 mo
animals. C. SOX2 nuclear quantification in ONLR and ON during
aging. SOX2 nuclei decline twofold from 1 mo-2 y (black bars) while
SOX2 nuclear numbers in distal ON (white bars) remain relatively
constant during this time (n=6 nerves/group; Data.+-.sem). D.
rq-PCR analysis of nestin mRNA expression in mouse ONLR (n=6 nerves
pooled/group). Nestin mRNA levels decline >40% in the first year
of life. Only one pooled prep was prepared for each age group:
there are no error bars. E-H: Analysis of human ONLR-nestin and
-SOX2 expression, with SOX2 immunostaining (green) and Nestin
(red). E. 7 month fetal ONLR. Strong SOX2 nuclear staining and
nestin expression is concentrated in the ONLR. F. 9 y/o ONLR.
Strong filamentous nestin staining is indicated (arrows). G. 40
y/o. Reduced nestin expression, but SOX2(+) nuclei are still
numerous. H. 50 y/o. Reduced filamentous nestin expression, with
remaining nestin present almost exclusively in the vasculature, and
greatly reduced SOX2(+) nuclei. Inset: 71 y/o ONLR. The elderly
ONLR reveals both a total lack of filamentous nestin and an absence
of SOX2(+) nuclei. I. Densitometric analysis of age-related human
ONLR nestin expression. Nestin expression declines linearly from 9
y/o to 45 y/o, with severe loss in individuals >50 y of age. The
inset shows donor history. J. Human ONLR neurosphere formation (33
y/o donor). K. SOX2/nestin expression in human neurospheres
generated from 3.sup.rd passage ONLR culture (33 y/o individual).
K. SOX2 (green)/nestin (red) Immunolabeling of human neurosphere
from culture shown in J. Individual cells express both SOX2 and
nestin L. Nestin (red)/GFAP (green) expression in human
neurosphere. Nestin and GFAP colocalize in individual cells. Scale
bar: 100 um.
DETAILED DESCRIPTION
I. Definitions
[0033] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found, for example, in Benjamin Lewin, Genes XII,
published by Jones & Bartlett Learning, 2014 (ISBN 1449659853);
Kendrew et al. (eds.); The Encyclopedia of Molecular Biology,
published by Blackwell Publishers, 1994 (ISBN 0632021829); and
Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a
Comprehensive Desk Reference, published by Wiley, John & Sons,
Inc., 1995 (ISBN 0471186341); and other similar technical
references.
[0034] As used herein, "a" or "an" may mean one or more. As used
herein when used in conjunction with the word "comprising," the
words "a" or "an" may mean one or more than one. As used herein
"another" may mean at least a second or more. Furthermore, unless
otherwise required by context, singular terms include pluralities
and plural terms include the singular.
[0035] As used herein, "about" refers to a numeric value,
including, for example, whole numbers, fractions, and percentages,
whether or not explicitly indicated. The term "about" generally
refers to a range of numerical values (e.g., +/-5-10% of the
recited value) that one of ordinary skill in the art would consider
equivalent to the recited value (e.g., having the same function or
result). In some instances, the term "about" may include numerical
values that are rounded to the nearest significant figure.
II. The Present Invention
[0036] A mitotically active, age-depletable population of neural
progenitor cells (NPC) has been discovered in the optic nerve
lamina region (ONLR) of the eyes of humans and rodents. As further
described herein, these ONLR-NPCs have a unique genetic signature
and they produce growth and survival factors thought to play a key
role in maintaining ocular health and function.
[0037] Because ONLR-NPCs are lost during aging, in glaucoma, and in
other optic nerve diseases, loss of ONLR-NPCs and the growth and
survival factors they produce are thought to contribute to optic
nerve diseases. Replacement of these secreted factors in the eye,
either alone or in combination with other treatments (e.g., those
that reduce ocular pressure), may serve to improve the treatment of
glaucoma and other optic nerve diseases.
[0038] As described herein, this population of ONLR-NPCs possess
properties typically found in NPCs. For example, ONLR-NPCs
co-express SOX1 and SOX2, as well as a number of NPC-specific
proteins including nestin, Glial Fibrillary Acidic Protein (GFAP)
and vimentin. ONLR-NPCs also express NPC-associated growth factors
such as brain derived neurotrophic factor (BDNF). Thus, the
postnatal ONLR-NPC niche is capable of generating both types of
glial cells in the ON, and enabling normal postnatal ON growth,
myelination, fluid regulation and cellular replacement. ONLR-NPCs
do not express Neural/Glial Antigen 2 (NG2).
Optic Nerve Lamina Region Neural Progenitor Cells (ONLR-NPCs)
[0039] The present invention is directed, in part, to optic nerve
lamina region neural progenitor cells (ONLR-NPCs) as defined
herein. ONLR-NPCs of the invention are characterized as cells
located in the ONLR of humans and rodents.
[0040] The ONLR-NPCs of the present invention can be defined based
on the expression, or lack thereof, of selected marker proteins.
The ONLR-NPCs of the present invention can thus be defined as
nestin(+), SOX2(+), GFAP(+), NG2(-) neural cells. SOX2 is the
transcription factor SRY (sex determining region Y)-box 2. GFAP is
glial fibrillary acidic protein. NG2 is neuron-glial antigen 2. In
some aspects of the invention, ONLR-NPCs also express one or more
of SOX1, vimentin, and BDNF. Thus, in certain aspects of the
invention, the ONLR-NPCs are:
[0041] nestin(+), SOX2(+), GFAP(+), NG2(-), SOX1(+); or
[0042] nestin(+), SOX2(+), GFAP(+), NG2(-), vimentin(+); or
[0043] nestin(+), SOX2(+), GFAP(+), NG2(-), BDNF(+); or
[0044] nestin(+), SOX2(+), GFAP(+), NG2(-), SOX1(+), vimentin(+);
or
[0045] nestin(+), SOX2(+), GFAP(+), NG2(-), SOX1(+), BDNF(+);
or
[0046] nestin(+), SOX2(+), GFAP(+), NG2(-), vimentin(+), BDNF(+);
or
[0047] nestin(+), SOX2(+), GFAP(+), NG2(-), SOX1(+), vimentin(+),
BDNF(+).
[0048] In each aspect of the invention, the cells are positionally
defined as residing within the ONLR, but exhibiting
nestin(+)/SOX2(+)/NG2(-) characteristics, unlike the cells in the
rest of the eye and optic nerve.
[0049] Alternatively, or in addition, the ONLR-NPCs of the
invention can be defined based on expression of one or more of the
secreted factors provided in Table 1. In one aspect of the
invention, the ONLR-NPCs of the present invention can be defined as
neural cells that secrete one or more of Latent Transforming Growth
Factor-Beta 1 (TGF-.beta.1), Connective Tissue Growth Factor
(CTGF), Nerve Growth Factor (NGF), Fibroblast Growth Factor
(FGF-1), Vascular Endothelial Growth Factor-A (VEGFA), Mesenchymal
Astrocyte-derived Neurotrophic Factor (MANF) and Insulin-like
Growth Factors-1 and -2 (IGF-1 and IGF-2). In a related aspect, the
ONLR-NPCs secrete each of Latent Transforming Growth Factor-beta 1
(TGF-.beta.1), Connective Tissue Growth Factor (CTGF), Nerve Growth
Factor (NGF), Fibroblast Growth Factor (FGF-1), Vascular
Endothelial Growth Factor-A (VEGFA), Mesenchymal Astrocyte-derived
Neurotrophic Factor (MANF) and Insulin-like Growth Factors-1 and -2
(IGF-1 and IGF-2).
[0050] The ONLR-NPCs of the present invention can also be defined
as neural cells that secrete at least 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16 or each of the growth factors provided in Table 1.
In one aspect, the ONLR-NPCs secrete each of the growth factors
provided in Table 1. In one aspect of the invention, the ONLR-NPCs
of the present invention are nestin(+), SOX2(+), GFAP(+), NG2(-)
neural cells that secrete at least 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16 or each of the growth factors provided in Table 1.
TABLE-US-00001 TABLE 1 Secreted factors produced by ONLR-NPCs VGF
Nerve Growth Factor inducible (VGF) Transforming Growth Factor,
beta-1, 68 kDa (TGF.beta.1) Latent Transforming Growth Factor
Binding Protein Beta 1 (LTBP1) Latent Transforming Growth Factor
Binding Protein Beta 2 (LTBP2) Platelet-derived Growth Factor beta
polypeptide (PDGFB) Connective Tissue Growth Factor (CTGF)
Fibroblast Growth Factor 11 (FGF11) Neuron-derived Neurotrophic
Factor (NDNF) Platelet-derived Growth Factor C (PDGFC) Transforming
Growth Factor, beta 2 (TGF.beta.2) Nerve Growth Factor (NGF)
Fibroblast Growth Factor 1 (FGF1) Midkine (Neurite Growth-promoting
Factor 2) Vascular Endothelial Growth Factor A (VEGFA)
Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF)
Insulin-like Growth Factors-1 and-2 (IGF-1 and IGF-2)
[0051] The ONLR-NPCs of the invention may be further characterized
as positionally isolated and residing within the ONLR (see FIGS. 3D
and 3E), but exhibiting nestin(+)/SOX2(+)/NG2(-) characteristics,
unlike the cells in the rest of the eye and optic nerve. ONLR-NPCs
of the invention may be additionally characterized as exhibiting an
extended `starlike` morphology in living animals (see FIGS. 3F and
3G).
[0052] The ONLR-NPCs of the invention may also be characterized
based on their ability to express at least 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15 or each of the proteins listed in Table 2.
TABLE-US-00002 TABLE 2 Additional proteins produced by ONLR-NPCs
Cell Growth Regulator with Ring Finger Domain 1 Dynein Regulatory
Complex Subunit 4 Fibroblast Growth Factor 12 Fibroblast Growth
Factor 14 Fibroblast Growth Factor Receptor 4 Growth Arrest and DNA
Damage-inducible Protein GADD45 beta Growth Factor Independent 1
Transcriptional Repressor Growth Hormone Receptor Placental Growth
Factor Platelet-derived Growth Factor D/Spinal Cord-Derived Growth
Factor B Platelet-derived Growth Factor Receptor-like Protein
Transforming Growth Factor beta-1 Proprotein Transforming Growth
Factor beta-1-induced Transcript 1 Protein Upper Zone of Growth
Plate and Cartilage Matrix Associated Protein Vascular Endothelial
Growth Factor C UNC-13 Homolog C
[0053] The present invention includes single ONLR-NPCs, populations
of ONLR-NPCs comprising two or more cells, and cultures of
ONLR-NPCs, i.e., in vitro cell cultures comprising ONLR-NPCs and in
vitro cell cultures consisting ONLR-NPCs.
ONLR-NPC Conditioned Media
[0054] The invention is also directed to ONLR-NPC conditioned
media. Such media is cell culture media in which ONLR-NPCs have
been grown, such as an in vitro cell culture of ONLR-NPCs. Because
ONLR-NPCs secrete factors, such as growth factors and survival
factors, the media in which the cells are grown becomes conditioned
by the presence of the secreted factors.
[0055] As used herein, ONLR-NPC conditioned media is any cell
culture media in which ONLR-NPCs have been present for at least 5
minutes. In some aspects, ONLR-NPC conditioned media is cell
culture media in which ONLR-NPCs have been present for at least 15,
30, 45, 60, 75, 90, 105 or 120 minutes, or 2.5, 3, 3.5, 4, 4.5, 5,
5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1.5, 2,
2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,
10.5, 11, 11.5 or 12 days, or more.
[0056] ONLR-NPCs are typically grown in Neurobasal.TM.
(ThermoFisher Scientific) medium supplemented with FGF-2 and -4,
and B27 supplementation, and grown on enhanced extracellular matrix
(matrigel) at concentrations typically exceeding 10% media.
However, the skilled artisan will understand that variations in the
identity and amounts of the components in such media can be made
without altering the health or growth the ONLR-NPCs, or the factors
secreted by the cells into the media. It should therefore be
understood that the identity of the media in which the cells are
cultured is not critical and that it can vary. In a preferred
aspect, the media does not include any animal sourced materials
(ASM) and is thus ASM-free.
[0057] ONLR-NPCs are typically grown under conditions of enriched
CO.sub.2 (5%), oxygen concentrations ranging from 5-21%, and 95%
relative humidity.
[0058] ONLR-NPC conditioned media may be collected from cultures of
ONLR-NPC through techniques well known in the art, such as simply
decanting the media into a separate vessel. ONLR-NPC conditioned
media may be stored, such as at 4.degree. C. or frozen, or used
immediately. ONLR-NPC conditioned media may be processed to
concentrate the media and/or to remove the secreted factors from
the media. Suitable processes include concentrating the decanted
media, following by dialysis and/or gel filtration to eliminate
both uninvolved medium, ions and large molecular weight compounds
such as bovine serum albumen.
ONLR-NPC Lysates
[0059] The invention is also directed to ONLR-NPC cellular lysates.
The ONLR-NPC lysates of the invention comprise all cellular
components from a single ONLR-NPC or from a population of
ONLR-NPCs.
[0060] ONLR-NPC lysates are produced by rupturing cellular
membranes and collecting all of the resulting materials. ONLR-NPC
lysates may be prepared by collecting the ONLR-NPCs grown in
appropriate medium through centrifugation, washing, and disruption
of the cells. Means for rupturing cellular membranes include
mechanical, ultrasonic, physical, enzymatic, chemical and osmotic
mechanisms. Mechanical mechanisms include repeated freeze/thaw
cycles, sonication, pressure, and filtration. Chemical mechanisms
include detergents such as Triton X-100. Osmotic mechanism include
subject the cells to hypo- or hyper-tonic environments. Physical
mechanisms include scraping and collecting cells from culture
plates.
ONLR-NPC Extracts
[0061] The invention is also directed to ONLR-NPC cellular
extracts. The ONLR-NPC extracts of the invention comprise less than
all of the cellular components from a single ONLR-NPC or from a
population of ONLR-NPCs.
[0062] ONLR-NPC extracts may be produced from ONLR-NPCs by first
rupturing cellular membranes, through such means as described
herein, and then collecting or isolating selected cellular
materials, such as one or more growth factors.
[0063] ONLR-NPC extracts may also be produced from ONLR-NPC
cultures by collecting conditioned media into which selected
cellular materials, such as growth factors, have been secreted and
then isolating selected cellular materials from the collected
media.
[0064] ONLR-NPC extracts may further be produced by preparing an
"artificial" ONLR-NPC extract that comprises selected cellular
materials, such as growth factors (e.g. one or more of the growth
factors listed in Table 1, i.e. VGF, TGF.beta.1, LTBP1, LTBP2,
PDGFB, CTGF, FGF11, NDNF, PDGFC, TGF.beta.2, NGF, FGF1, Midkine,
VEGFA, MANF, IGF-1, and IGF-2), produced by means other than the
ONLR-NPCs, such as by collecting the cellular materials from media
of cells engineered to produce the selected cellular materials. The
"artificial" ONLR-NPC extracts of the invention include (i) those
having one or more of the growth factors of Table 1, (ii) those
having one or more of the proteins of Table 2, and (iii) those
having one or more of the growth factors of Table 1 and those
having one or more of the proteins of Table 2.
[0065] Means for producing and isolating the cellular materials are
well known to the skilled artisan and will vary depending on the
identity of the cellular material(s) to be collected or isolated,
and the manner in which they are produced. However, suitable means
include centrifugation, filters and columns to screen materials
based on size, antibody coated chips, plates and columns to isolate
peptides/proteins, for example, based on binding affinity.
Compositions Comprising Factors Secreted by ONLR-NPCs
[0066] As indicated above, the ONLR-NPCs of the invention can be
defined based on expression of one or more of the secreted factors
provided in Table 1 and/or expression of one or more of the
proteins provided in Table 2. While the factors will be known the
one skilled in the art, the present invention also includes (i)
compositions comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16 or each of the secreted factors provided in Table 1, (ii)
compositions comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16 or each of the proteins provided in Table 2, and (iii)
compositions comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16 or each of the secreted factors provided in Table 1 and at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or each of the
proteins provided in Table 2.
Methods of Treatment
[0067] The invention is also directed to methods of treating or
preventing optic nerve diseases. Because ONLR-NPCs are thought to
play a role in supporting glial cell growth and development, and by
extension, myelination of axons as they emerge from the eye,
ONLR-NPCs and the factors they secrete are likely to have activity
in treating or preventing optic nerve hypoplasia, regional axonal
dysfunction and hypomyelination. ONLR-NPCs and the factors they
secrete may also enable glial cell replacement and
remyelination.
[0068] The methods of treating or preventing optic nerve diseases
of the invention comprise administering to a subject in need
thereof a therapeutically effective amount of a composition
comprising one or more of:
[0069] (i) a population of ONLR-NPCs,
[0070] (ii) ONLR-NPC conditioned media,
[0071] (iii) an ONLR-NPC lysate,
[0072] (iv) an ONLR-NPC extract, and
[0073] (v) one or more factors secreted by ONLR-NPCs,
thereby treating or preventing an optic nerve disease in the
subject.
[0074] Optic nerve diseases that may be treated using the methods
of the present invention include, but are not limited to,
open-angle glaucoma, angle-closure glaucoma, optic nerve
hypoplasia, optic nerve hypomyelination, regional axonal
dysfunction, nonarteritic anterior ischemic optic neuropathy
(NAION), and optic neuritis.
[0075] In one aspect, the composition comprises one or more factors
secreted by ONLR-NPCs, wherein the factors are selected from the
group consisting of Nerve Growth Factor (NGF), Latent Transforming
Growth Factor-Beta 1 (TGF-.beta.1), Fibroblast Growth Factor 1
(FGF1), Vascular Endothelial Growth Factor (VEGF), Mesenchymal
Astrocyte Neurotrophic Factor (MANF), Connective Tissue Growth
Factor (CTGF), and Insulin-like Growth Factors-1 and -2 (IGF-1 and
IGF-2). In another aspect, the composition comprises one or more of
the proteins produced by ONLR-NPCs listed in Table 2. In further
aspect, the composition comprises one or more of the secreted
factors of Table 1 and one or more of the proteins of Table 2.
[0076] In each of the methods of the present invention, a
"therapeutically effective amount" of a composition comprising one
or more of (i) a population of ONLR-NPCs, (ii) ONLR-NPC conditioned
media, (iii) an ONLR-NPC lysate, (iv) an ONLR-NPC extract, and (v)
one or more factors secreted by ONLR-NPCs is administered to a
subject. The effective amount will vary between subjects and the
identity of the optic nerve disease being treated. However, the
effective amount is one that is sufficient to achieve the aim or
goal of the method.
[0077] In the methods of the invention, the composition may be
administered to the eye of the subject, such as via topical
application to the surface of the eye (e.g. eye drops), via
subconjunctival injection, via intravitreal injection into the
interior of the eye, or via retrobulbar injection into the space
behind the globe of the eye. Such injections include depot
injection through the conjunctiva.
[0078] Administration of the composition may be via any of the
means commonly known in the art. When administered to the eye, such
routes include topical and intraocular, subconjunctival and
retrobulbar injections. When administered to sites other than the
eye, such routes include intravenous, intraperitoneal,
intramuscular, subcutaneous and intradermal routes of
administration, as well as topical, nasal application, by
inhalation, orally, rectally, vaginally, or by any other suitable
mode.
[0079] The pharmaceutical compositions of the present invention may
be formulated, for example, for topical, intraocular, oral,
sublingual, intranasal, rectal, transdermal, mucosal, pulmonary, or
parenteral administration. Parenteral modes of administration
include without limitation, intradermal, subcutaneous (s.c., s.q.,
sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.),
intraperitoneal (i.p.), intra-arterial, intramedulary,
intracardiac, intra-articular (joint), intrasynovial (joint fluid
area), intracranial, intraspinal, and intrathecal (spinal fluids).
Any known device useful for parenteral injection or infusion of
drug formulations can be used to effect such administration. In
preferred aspects on the invention, the composition is administered
to the subject as a topical drop for ophthalmic administration.
[0080] Appropriate doses and dosing schedules can readily be
determined by an attending physician without undue experimentation
depending on the characteristics of the subject to be treated and
the identity of the optic nerve disease. Administration frequencies
for the compositions of the present invention include 4, 3, 2 or
once daily, every other day, every third day, every fourth day,
every fifth day, every sixth day, once weekly, every eight days,
every nine days, every ten days, bi-weekly, monthly and bi-monthly.
The duration of treatment will be based on the optic nerve disease
being treated and will be best determined by the attending
physician.
[0081] In the methods of the invention, the composition
administered to the subject may be in the form of a pharmaceutical
formulation comprising (a) one or more of (i) a population of
ONLR-NPCs, (ii) ONLR-NPC conditioned media, (iii) an ONLR-NPC
lysate, (iv) an ONLR-NPC extract, and (v) one or more factors
secreted by ONLR-NPCs and (b) a pharmaceutically acceptable
carrier.
[0082] Suitable examples of carriers are well known to those
skilled in the art and include water, water-for-injection, saline,
buffered saline, dextrose, glycerol, ethanol, propylene glycol,
polysorbate 80 (Tween-80.TM.), poly(ethylene)glycol 300 and 400
(PEG 300 and 400), PEGylated castor oil (e.g. Cremophor EL),
poloxamer 407 and 188, hydrophilic and hydrophobic carriers, and
combinations thereof. Hydrophobic carriers include, for example,
fat emulsions, lipids, PEGylated phospholipids, polymer matrices,
biocompatible polymers, lipospheres, vesicles, particles, and
liposomes. The terms specifically exclude cell culture medium. The
formulations may further comprise stabilizing agents, buffers,
antioxidants and preservatives, tonicity agents, bulking agents,
emulsifiers, suspending or viscosity agents, inert diluents,
fillers, and combinations thereof.
[0083] The subject may be a human, a non-human primate, bird,
horse, cow, goat, sheep, a companion animal, such as a dog, cat or
rodent, or other mammal.
III. Examples
A. Experimental Protocols
Mouse Optic Nerve (ON) and Optic Nerve Lamina Region (ONLR)
Dissection for Immunohistochemistry (IHC)
[0084] ON and ONLR used for IHC were isolated from mice perfused
with 4% paraformaldehyde-phosphate buffered saline (PFA), by
enucleating eye globes, leaving at least 3 mm of attached ON. The
remaining ON was collected following craniotomy, removing the ON
and adjacent optic chiasm. Tissues were then placed in 2% PFA,
overnight, rinsed and transferred to 30% sucrose for sectioning.
Tissue was embedded in optimal temperature cutting solution (OCT),
frozen at -80.degree. C.; and 10 um cross- or longitudinal sections
were cut using Leica cryostat.
Immunohistochemistry (IHC) Procedure
[0085] Sections were subjected to antigen retrieval procedure
following fixation. Briefly, sections were incubated in 10 mM
sodium citrate buffer for 10 min at 80.degree. C., rinsed and
blocked with 2% normal donkey serum for 1 hr, incubated with the
primary antibody; see antibody list in Table 4) overnight at
4.degree. C. Sections were then rinsed in PBS containing 0.5%
Triton X100, incubated with appropriate secondary antibody for one
hour at 25.degree. C., counterstained with DAPI, mounted on a slide
with appropriate medium and stored at 4.degree. C. until use.
Dissection and Culture of Mouse Lamina and ON Tissue Neural
Progenitor Cells (NPCs)
[0086] The globes and attached ONs of appropriately aged (typically
17-18 d) mice were enucleated, leaving at least a 3 mm stump of ON
attached. Distal ON was obtained separately, measured from the
distal stump. Typically, at least 10 laminae were collected for NPC
isolation. Tissues were then placed into a 100 mm culture dish with
cold Hibernate-A.RTM. (GIBCO A12475-01) until dissociation, and the
medium changed immediately prior to dissociation.
[0087] ONLRs were isolated from the globe by trimming adjacent ON
to 3 mm in a laminar flow hood. A small hole was made at the base
of the globe 1 mm from the ON stump using a 29 ga needle. Vannas
scissors were used to dissect around the ONLR and adjacent ON. The
remaining sclera was removed, leaving only the ON and sheath
adjacent to the nerve, as it inserts into the globe. ONLR sections
were trimmed to 800 microns distally. The ON sheath was carefully
dissected from the ONLR by rolling back the sheath with a 29 gauge
needle-knife and Vannas scissors, and isolated ONLR tissues were
placed into ice-cold Hibernate-A.RTM. media. Because of the small
size, mouse ONLRs were not further triturated (unlike human, which
were triturated using 2-29 ga needles as knives). Two mm of distal
ON, separated from the ONLR by at least 2 mm, were isolated in a
similar manner and dissected into smaller pieces using 29 ga needle
knives. Tissues were placed into a 1.5 ml micro-centrifuge tube
containing 300 ul of Hibernate-A.RTM. media and kept on ice.
ONLR-NPC and ON Cell Cultivation
[0088] Plates were coated with 15% Matrigel.RTM. (VWR/Corning, cat
#354234) diluted in DMEM for a minimum of one hour prior to cell
addition at room temperature. Immediately before cell plating,
unbound Matrigel was carefully aspirated, and 200 ul of complete
NT2 medium (see below) was added per well of a 24-well plate (cat
no. 662102 CELLSTAR.RTM.).
[0089] Briefly, ON/ONLR tissues were centrifuged at 300.times.g for
3 min. Hibernate-A.RTM. media was discarded and replaced with
Dulbeccos complete phosphate buffered saline (DPBS) containing
penicillin/streptomycin (GIBCO cat #15140-122). The ON/ONLR tissues
were placed on ice for 5 min, then centrifuged at 300.times.g for 3
min. The DPBS solution was discarded, and the ON/ONLR tissue was
washed twice with fresh DPBS.
[0090] 300 ul of collagenase (1 mg/ml in sterile DPBS; GIBCO cat
#17104-019) was added to the tissues, and these were incubated at
37.degree. C., for 10-15 mins. The latter time was used depending
on the degree of tissue digestion, to eliminate residual undigested
tissues. Solutions were aspirated 10-15 times at 10 minutes using a
p1000 pipette to homogenize tissues, avoiding air bubbles from
forming. Tissue was placed for a further 5 min at 37.degree. C. for
digestion if required. An equal volume of complete NT2 medium (see
composition below in Table 3) was then added to the tubes,
centrifuged at 300.times.g for 5 min, and the supernatant
(containing collagenase) was discarded. The resulting pellet was
resuspended 5 times using a p1000 pipettor, to enable even cell
distribution into 1 ml of media, containing 0.5 ul/ml of fungizone
(GIBCO cat #15290-018), and an aliquot of the dissociated cells
were counted manually using a hematocytometer.
[0091] Cells were plated according to cell density. Up to two
additional volumes of medium were added if the bottom of the cell
plate was not visible in the Matrigel.RTM. coated 24-well plates.
Cells were then placed into a humidity controlled incubator at
37.degree. C. supplemented with 5% CO.sub.2. Final volume in each
well was .about.500 ul. The cells were then left untouched for five
days prior to initial feeding to enable cell adherence to the plate
prior to addition of new media. Wells were supplemented with 250
ul/well additional fresh medium. Cells were subsequently fed every
2-3 days thereafter with complete NT2 medium. 1/2 of the old medium
(250 ul) was removed during each subsequent feeding. Medium was
prepared fresh every 2 weeks. Matrigel was applied to the well one
hour prior to cell reseeding.
TABLE-US-00003 TABLE 3 Complete NT2 media composition Component
Company Cat # Final Concentration Neurobasal Invitrogen 21103049
48% DMEM/F12 Invitrogen 11330032 48% B27-50x Invitrogen 12587010 1%
NC-N2 suppl Invitrogen 17502048 0.5% Insulin Sigma-Aldrich 05165
0.1% BSA MP Biomedicals 199896 0.5 mg/ml FGF-2 R&D Systems
233-FB 10 ng/ml FGF-4 R&D Systems 235-F4 10 ng/ml Gentamicin
Invitrogen 15750060 2 .mu.g/ml
Immunocytochemistry (ICC) of Cultured Cells
[0092] Plated cells used for ICC were aspirated and rinsed twice
with DPBS and fixed in 4% PFA for 10 min. Cells were subsequently
rinsed twice with DPBS-0.05% Triton (DPBST) for 10 min and
incubated with primary antibody (see list of primary antibodies in
Table 4) in 5% (Donkey Normal Serum) at 4.degree. C. overnight.
Following two additional rinses with DPBST, cells were incubated
with appropriate secondary antibodies for 1 hour at RT (1:500
dilution in DPBST-5% normal serum). Afterwards cells were rinsed
twice with DPBS (5 min), counterstained with DAPI (1:10K), mounted
with fluorescence enhancing media and observed at confocal Zeiss
LSM 510 microscope.
TABLE-US-00004 TABLE 4 Antibody Type Company Catalog # Anti-NG2
Chondroitin Monoclonal Millipore MAB5384 sulfate proteoglycan
Anti-Nestin (rat/mouse) Monoclonal Millipore MAB353 clone 401
Anti-Nestin (human) Polyclonal Sigma N5413 Anti-SOX2 Polyclonal
Abcam ab97959 (mouse/human) Anti-SOX2 Monoclonal Thermo-Fisher
20G5/ (human/mouse) MA1-014 Anti-SOX1 Polyclonal Cell Signaling
#4194 (human/mouse) Anti-NeuN Monoclonal Millipore MAB377
(mouse/human) Anti-TUJ1 Monoclonal DHSB 6G7 (human/mouse) A2B5
(human/mouse) Monoclonal Abcam ab53521 Olig-1 Monoclonal Neuromab
N149/25 GFP ms Polyclonal Invitrogen A11122 GFP ck Polyclonal
Millipore #06-896 AQP4 (human/mouse) Polyclonal Alomone AQP-014
GFAP (human/mouse) Monoclonal Abcam GF5/ ab10062 GFAP (human/mouse)
Polyclonal Dako Z0334 MBP (human/mouse) Polyclonal Sigma-Ald M3821
O.sub.4 (human/mouse) Monoclonal Millipore MAB345 Ki67
(human/mouse) Rbt monocle Abcam ab16667 PAX6 (chicken) Monoclonal
DHSB P3U1/ ab528427 PAX2 (human) Polyclonal Abcam AB38738 SCA-1
(mouse) Polyclonal Millipore AB4336 STRO-1 (human) Monoclonal
Millipore MAB4315
Formation and Immunostaining of Neurospheres from Mouse
ON/Lamina/SVZ Cultured Cells
[0093] Formation of neurospheres (NS) was induced from second
passage mouse ON/ONLR/SVZ stem cells growing on Matrigel.RTM.
following growth to approximately 70% confluency. Half of the NT2
media was removed. Cells were carefully detached using a cell
scraper and transferred using a p1000 pipette to a new 50 mL tube.
An equal volume of fresh NT2 media was added to the tube and the
cells were re-distributed evenly in the media by gentle aspiration.
The cells were then transferred to an Ultra-Low Attachment Surface
6-well Plate (CORNING cat #3471 Costar.RTM.). 500 ul of NT2 media
was added to the wells prior to the cell seeding. Fresh NT2 media
was added every 2-3 days. NS formation was observed between 4-7
days. NS were counted and imaged. For IHC, NS were collected from
the plates and allowed to settle at the bottom of an Eppendorf
tube. The excess media was removed and NS were rinsed with DPBS,
pipetted gently to ensure even distribution and fixed in 2% PFA for
30 min at 4.degree. C. Subsequently NS were rinsed in 10 mM glycine
for 10 min with gentle shaking to prevent clumping, and collected
at 300.times.g for 2 min. Excess glycine was removed and
neurospheres were placed in 5% sucrose for 1 hr at 4.degree. C.,
cryoprotected in 30% sucrose, fixed in OCT at -80.degree. C. and
sectioned at 10 um.
In Vivo Transgenic Animal Studies
[0094] All experimental protocols and procedures with mice were
approved by Institutional Animal Care and Utilization Committee
(IACUC; Baltimore, Md., USA). Mice (both genders) were housed in
polycarbonate cages with density of 5 mice/cage on a 12-h
light/dark cycle. Mice had access to food and water ad libitum.
Animals were mated to corresponding strains and tail DNA analyzed
using the appropriate PCR primers to ensure appropriate
genotype(s). Non-mutant mice of the same background were used as
controls.
[0095] B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J mice
were crossed with B6; 129S-Sox2tm1(cre/ERT2)Hoch/J. Appropriately
identified genotyped double-mutant progeny were used for
experimental procedures. These were Tm1(cre/ERT2)Hoch-Sox2/J) X
Gt(ROSA) 26Sortm td-Tomato,GFP)Luo/JO, and referred as Sox-GFP
mice.
[0096] B6.129S-Sox2tm1(cre/ERT2)Hoch/J mice were crossed with
B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J and double mutants were used
for experimental procedures. The double mutants are referred as
Sox-DTA mice.
Tamoxifen and 4-Hydroxytamoxifen (4OHT) Injections
[0097] Tamoxifen or 4OHT injections (intraperitoneal (ip) and
local-retrobulbar (RB), respectively) were used to induce Cre
recombination. 4OHT was used for local injections because Tamoxifen
is a prodrug that needs to be metabolized by the liver to 4OHT
which itself is the active form. 30 mg of Tamoxifen (Sigma, cat
#5648) was moistened in 100 ul of 100% ethanol, vortexed for 30
sec, and 900 ul of sunflower seed oil (Sigma, cat #5007) was added.
The mixture was revortexed, sonicated for 2 min on ice. 80 ul of
Tamoxifen was subsequently injected ip (.about.15-20 mg mouse) four
times over 5 days. Retrobulbar 4OHT injections (Cayman, cat #17308)
were prepared as a 30 mg/ml suspension in 10% ethanol and 90%
ethoxylated castor oil (Kolliphor: Sigma, cat #5135). Five
microliters of this mixture was injected behind the eye (RB
injection).
Injection and Tissue Harvesting Schedule: Sox-GFP Mice
[0098] Tm1(cre/ERT2)Hoch-Sox2/J) X Gt(ROSA) 26Sortm
td-Tomato,GFP)Luo/JO injections were performed either ip or locally
(RB), according to the following times. Postnatal: 19 d
injection/all sexes; euthanized 14 d, 30 d and 60 d post-injection.
Adult: injection at 60 d PN/all sexes; euthanized at 14 d or 30 d
post-injection. Adult/aged: 6 mo, 1 y, 2 y ON and ONLR cell
culture: tissues were collected postmortem from perfused animals
following terminal anesthesia.
SOX2-DTA Mice
[0099] These were double mutants consisting of
Tm1(cre/ERT2)Hoch-Sox2/J) X Gt(ROSA)26Sortm1(DTA)Lkyb. It was
determined that i.p. Tamoxifen injections were routinely fatal
within 10 days, likely due to global stem cell suppression. For
this reason, local (RB) 4OHT administration was utilized. The local
approach had the added advantage of enabling the use of the
contralateral eye of each animal as its own vehicle control.
Animals were injected on one side with 4OHT, and the contralateral
side with vehicle (Kolliphor).
[0100] Postnatal: 19 d RB injection/all sexes; euthanized at 3 d,
14 d or 30 d post-injection Adult: 60 d injection/all sexes;
Animals were euthanized at 14 d or 30 d post-injection
Human ONLR Dissections and Stem Cell Cultivation
[0101] Fixed human tissues used for immunohistochemistry were
obtained from a commercial donor source (Lions Eye Institute for
Transplant and Research, Tampa, Fla.), following institutional
review board (IRB) exemption. Human tissue used for cultivation was
obtained from freshly isolated post-surgical specimens following
enucleation for severe, irreparable damage without hope of
recovery, and to prevent development of post-damage autoimmune
response to the contralateral eye (sympathetic ophthalmia).
Specimens were obtained following University of Maryland Medical
Center IRB approval. Eye tissues used for this purpose was stripped
of identifiable information except for sex. Tissues used for
ONLR-NPC cultivation were taken directly from the operating room on
ice, with maximum times from enucleation to final cultivation of 3
hours post enucleation.
[0102] To minimize contamination, enucleated tissues were initially
dipped twice in 5% povidone iodine-DPBS solution, and washed twice
in sterile DPBS before dissection. The optic nerve and adjacent
lamina entering the globe was located and carefully cut away from
the globe, using a sterile Vannas scissors. 5 mm of adjacent nerve
emerging from the globe was used for laminar isolation. The optic
nerve sheath was removed using the Vannas scissors, as was the
scleral wall surrounding the lamina as it emerges from the globe.
The anterior 2 mm of the retina entering the scleral wall was
excised. The intact ONLR was placed onto a 6-well plate with 1 mL
of ice-cold sterile Hibernate-A.RTM. media. The isolated ONLR
tissue was triturated using Vannas scissors, and tissue transferred
into a sterile conical tube and centrifuged at 300 g for 5 min.
Hibernate-A.RTM. media was discarded and replaced with DPBS
containing penicillin/streptomycin (GIBCO cat #15140-122). The ONLR
tissue was placed on ice for 5 min. It was then centrifuged at
300.times.g for 3 min, the DPBS solution discarded, and the ONLR
tissue washed twice with DPBS. One mL of collagenase (1 mg/ml in
sterile DPBS GIBCO cat #17104-019) was added to the tissue, and
manual dissociation of the tissue was used to break up larger
pieces. It was then placed into the incubator for activation, at
37.degree. C., for 15-20 mins (dependent on state of tissue
digestion).
[0103] A p1000 pipette (with a large bore tip) was used to further
dissociate the tissue, by aspirating 20-30 times carefully to avoid
formation of air bubbles, and then the tissue was placed for a
further 5-10 min at 37.degree. C. for digestion. An equivalent
volume of complete NT2 medium was added to the tube, centrifuged at
300.times.g for 5 min, and the supernatant discarded. The pellet
was evenly resuspended in 2 ml of NT2 medium containing 0.5 ul/ml
of fungizone (GIBCO cat #15290-018) using a p1000 pipette. Cells
were plated onto 15% Matrigel.RTM. coated 6-well plates and placed
into a humidity controlled incubator at 37.degree. C. The cells
were then left unmolested for three days to allow the cells to
adhere to the plate before the addition of new media.
Human ONLR Neurosphere Formation
[0104] Human ONLR cells were grown to 70% confluence and then
transferred onto new Matrigel.RTM. following each subculture (SC).
After SC2 human ONLR cells were transferred onto an Ultra-Low
Attachment Surface 10 cm Plate (CORNING cat #3262). Half of the NT2
media from the well was removed from the well. Cells were carefully
detached using a cell scraper and transferred using a 5 mL
serological pipette to a new 50 mL tube. An equal volume of fresh
NT2 media was added to the tube and the cells were re-distributed
evenly in the media by gentle pipetting up and down using a 5 mL
serological pipette. The cells were then transferred to an
Ultra-Low Attachment Surface 10 cm plate (CORNING cat #3262). 5 mL
of NT2 media was added to the wells prior to the cells being
plated. Fresh NT2 media was added every 2-3 days. NS formation took
between 7-14 days, upon which NS were counted and imaged. The ONLR
NS were also fixed, sectioned and stained.
RNA Isolation from Eye Tissues and Cultures
[0105] Because the ONLR region has such a high concentration of
extracellular matrix, the standard procedure was modified by
combining two RNA isolation procedures: Phenol/Guanidinium (RNAbee:
Tel-Test Cat #CS-104B). Initial RNA isolation was followed by
genomic DNA removal and further RNA clean up procedure using the
Qiagen (RNeasy micro kit, Cat #74004). This combination enhanced
carbohydrate removal, and improved RNA yields appreciably. ONLR and
ON RNA was isolated from a minimum of 10 pooled samples (5
animals/prep). Total RNA was isolated identically from retina, ONLR
and optic nerve. Total RNA was evaluated for yield and A260/A280
ratios using nanodrop, and for quality using an Agilent RNA Nano
electropherogram. Samples with RNA integrity number (RIN) below 8
were discarded.
Real Time Qualitative Polymerase Chain Reaction (RT-PCR)
[0106] First strand cDNA synthesis was performed using Invitrogen
SuperScript First-Strand synthesis system (Cat #11904-018) or
Superscript III (Cat #18080-051) with random primers. qPCR was
performed using iQ SYBR Green Supermix from Bio-Rad (Cat #170-8882)
on an iCycler (Biorad). The 15 ul qPCR reaction contained
1.times.SYBR Green Supermix, 400 nM gene specific primers and
various amounts of first strand cDNA, which was diluted to 1:10 in
water depend on the abundance of the gene tested. The gene specific
primers were obtained from IDT (Integrated DNA Technologies)
pre-designed primers or custom designed using the "Pick Primers"
program on the NCBI website. The parameters were set to have a
product size of less than 200 bp, 58-61.degree. C. annealing
temperature and crossed exon boundaries for forward and reverse
primers, to further reduce the chance of results skewed by genomic
contamination (sees Table 5 and 6 for detailed information). The
optimal primer pair and amount of cDNA used in the reaction were
also determined by the standard curve, which was required to yield
better than 90% amplification efficiency. qPCR reactions were
performed on the following parameter: 95.degree. C. 5 minutes for
first denaturation, followed by 40 cycles of denaturation at
95.degree. C. for 30 sec, annealing at 60.degree. C. for 30 sec,
and extension at 72.degree. C. for 30 sec, and a final 5 min
extension. Reactions which yielded single melting curve were used
in the quantitative analysis. The .DELTA..DELTA.Ct. method of
comparing relative sequence abundance was utilized, employing two
internal control genes for quality, consistent with MIQE
guidelines. All assays were run in duplicate.
TABLE-US-00005 Nucleotide primers used for gene expression Gene
symbol Product Cell Sequence Size Tnt type Acc# 5'-3' Polarity (bp)
.degree. C. Nes NM_ GAGGCGCTGG Plus 75 60 NPC 016701 AACAGAGATT
Minus (SEQ ID NO: 1) CACAGCCAGC TGGAACTTTTC (SEQ ID NO: 2) SOX1 NM_
GGCAGTCATAC Plus 104 60 NPC 009233 AAAAGTTGGC Minus (SEQ ID NO: 3)
GTACAGTATTT ATCGTCCGCAG A (SEQ ID NO: 4) SOX2 NM_ CAGGAGTTGT Plus
133 60 NPC 011443 CAAGGCAGAG Minus A (SEQ ID NO: 5) CTTAAGCCTC
GGGCTCCAAA (SEQ ID NO: 6) CSPG4/NG2 NM_ GACCAACCCC Plus 108 60 OPC
139001 CGBGGCGCAC Minus (SEQ ID NO: 7) TGGGCCCGAA TCATTGTCTG (SEQ
ID NO: 8) ALDH1L1 NM_ GCCTTGTTGA Plus 117 60 NPC/Astro 027406
TATCCCGAGT Minus (SEQ ID NO: 9) GCCCATCATG ATCATCTCTC G (SEQ ID NO:
10) Gli1 NM_ TGTGTAAGCA Plus 130 60 Hedgehog 010296 GAGCTCATGC
Minus effector (SEQ ID NO: 11) CATAGGGTCT CGGGGTCT (SEQ ID NO: 12)
Msi1 NM_ TGAGAGGGAT Plus 140 60 NPC 008629 AGCTGTGAGC Minus (SEQ ID
NO: 13) GTTCCAAGCC ACGACCTAC (SEQ ID NO: 14) PAX6 NM_ CACGTACAGT
Plus 134 60 Early 013627 GCTTTGCCAC Minus Neuron (SEQ ID NO: 15)
AACTCCGCCC ATTCACTGAC (SEQ ID NO: 16) Shh NM_ CGTAAGTCCT Plus 131
60 Adult 009170 TCACCAGCTT Minus NSCs G (SEQ ID NO: 17) GAATCCAAAG
CTCACATCCA C (SEQ ID NO: 18) Ppib NM_ GAGACTTCAC Plus 193 60
Reference 011149 CAGGGGAGAT Minus gene (SEQ ID NO: 19) AAAACCACAT
GCTTGCCATC C (SEQ ID NO: 20) GAPDH NM_ CAGCAAGGAC Plus 104 60
Reference 00804 ACTGAGCAAG Minus gene A (SEQ ID NO: 21) GCCCCTCCTG
TTATTATGGGG (SEQ ID NO: 22)
TABLE-US-00006 TABLE 6 Nucleotide primers used for genotyping
Sequence Product Genotype 5`-3` Tann (bp) ER2-SOX2-Cre GCGGTCTGGCA
51.7.degree. C. 100 GTAAAACTATC (SEQ ID NO: 23) GTGAAACAGCA
TTGCTGTCACT T (SEQ ID NO: 24) ROSA-DTA mt CGACCTGCAGG 65.degree. C.
650 TCCTCG (SEQ ID NO:25) CTCGAGTTTGT CCAATTATGTC AC (SEQ ID NO:
26) ROSA-DTA wt CCAAAGTCGCT 65.degree. C. 604 CTGAGTTGTTA TC (SEQ
ID NO: 27) GAGCGGGAGAA ATGGATATG (SEQ ID NO: 28) Td-Tom common
CTCTGCTGCCT 62.degree. C. 575 CCTGGCTTCT (SEQ ID NO: 29) Td-Tomato
mt TCAATGGGCGG 69.degree. C. 250 GGGTCGTT (SEQ ID NO: 30) Td-Tomato
wt CGAGGCGGATC 62.degree. C. 330 ACAAGCAATA (SEQ ID NO: 31)
Vascular Imaging Using Fluorescein-Linked Bovine Serum Albumin
(FITC-BSA).
[0107] FITC-BSA was prepared and dissolved in porcine gelatin
(Sigma-Aldrich) as previously described (Nicholson et al, 2012).
Terminally anesthetized animals were transcardially-perfused with
the dissolved reagent after clamping the descending aorta and
allowing for continuous flow through the opened right atrium. The
neck vasculature was clamped following perfusion, and the animal
immersed in an ice water bath for 30 minutes to set the gelatin.
The eyes and adjacent ON were dissected and post-fixed in 4% PF-PBS
for three days. ONLR and ONs were dissected as previously
described, and the tissue imaged using a Zeiss two-photon
microscope to a depth of 500 um.
Transmission Electron Microscopy
[0108] Terminally anesthetized mice were transcardially-perfused
with 2% PF-PBS and ocular tissues isolated and post-fixed in
buffered fixative containing paraformaldehyde and glutaraldehyde.
Tissues were stained with uranyl acetate and embedded in Epon, and
cross-sectioned at 200 nm. The anterior ON region was evaluated 1
mm from the globe (avoiding the ONLR), and the distal ON 4 mm from
the globe, using a Tecnai transmission electron microscope at
2100.times. and 4400.times.. Quantification of the differences in
relative axonal myelination was performed by counting axons using
5-2100.times. micrographs per optic nerve.
ON-Myelin Thickness Ratio (Q-Ratio)
[0109] Q-ratios were evaluated from five cross sections/optic nerve
at 4400.times. using quantification of individual axons provided by
Image J. The inner (unmyelinated) and outer (myelinated) area of
each axon was defined using the freehand outline-polygon function,
and each area was converted to their circular equivalents. The
ratio of inner/outer radius for each axon was determined; data from
axons were pooled into very small (<400 nm), small (400-600 nm)
medium (600-800 nm), large (800-1000 nm), very large (1000-1200
nm), and giant (>1220 nm) axons.
B. ONLR Structure is Consistent with an NPC Niche.
[0110] The mammalian ONLR in humans and rodents presents as a
narrow band of unmyelinated tissue directly adjacent to the retina
(FIG. 1B). The mouse ONLR's cellular organization was examined,
with its boundaries defined by an absence of myelin, using the
oligodendrocyte- and OPC-specific O.sub.4 antibody (Table 4) (FIG.
1D, in green). Expression of the NPC-expressed intermediate
filament protein nestin was also evaluated (Park et al., 2010).
Filamentous nestin expression in the ON is localized to the
unmyelinated ONLR (FIG. 1D, in red). Glial fibrillary acidic
protein (GFAP), another adult NPC marker (Gorris et al., 2015), is
co-expressed by the majority of ONLR-nestin expressing cells (FIG.
1F). The water channel protein aquaporin 4 (AQP4) is expressed at
high levels in astrocytes (Cavazzin et al., 2006), and at reduced
levels in NPC niche sites (Tavazoie et al., 2008). Confocal
analysis revealed that ONLR-AQP4 expression levels are reduced
(FIG. 1E, in green), despite the abundant presence of astrocytes,
compared with either retina or distal ON (FIG. 1E, Retina and
ON).
[0111] Whether cells in the ONLR express SOX2 protein, another
hallmark of NPCs, was examined. Many cells in the ONLR that express
both filamentous nestin and SOX2 were found (FIG. 1G, arrowheads),
while nearly all cells in the distal ON do not (inset, FIG. 1G).
SOX2-expressing cells make up nearly 50% of all nuclei in the 60 d
mouse lamina (FIG. 1H, 247.+-.31.8 SOX2(+) nuclei/ONLR v.
486.+-.41.6 total ONLR nuclei, n=3 eyes; .+-.sem), while comprising
only 17% of nuclei in the mid-ON region (FIG. 1H; 207.+-.17.9
SOX2(+) nuclei/ON v. 1193.+-.127 nuclei/ON, n=3; .+-.sem).
Nestin/GFAP expression is lost in the distal ON (FIGS. 1 G and J
insets). ONLR-SOX2-expressing cells also express GFAP (FIG. 1J).
Hence, the ONLR includes numerous cells co-expressing these three
characteristic markers of adult NPCs.
[0112] To determine ONLR cell proliferation, in vivo mitotic
activity was quantified using Ki67 antibody (Table 4) in 60 d
(adult) ONLR, and compared to the distal ON region (FIG. 1I). The
ONLR contains .about.6.2 times as many mitotic nuclei (per unit
nuclei) and 3.5-fold higher mitotic nuclei (per unit area) than the
distal ON (inset, Fig I, N=3 eyes/group). An increased mitotic
index of ONLR-NPCs relative to surrounding tissue is consistent
with their role in adult neural cell replacement (Stoll et al.,
2011).
[0113] NPCs in the adult CNS in vivo give rise to neurons as well
as astrocyte and oligodendrocyte progeny (Ahlenius et al., 2009).
However, no expression of the early neuronal markers doublecortin,
PAX6 or NeuN was found in the ONLR (data not shown). SOX2(+) ONLR
cells are NG2(-) (FIG. 1K, ONL and also compare ONL and ON in FIG.
1L). NG2(+)/nestin(-) OPCs were detectable as a band of cells in
the area immediately below the ONLR. Their location adjacent to the
ONLR is consistent with possible derivation from ONLR-NPCs (FIG. 1K
and inset; also FIG. 1L, arrows). These results suggest that
ONLR-resident SOX2 expressing cells constitute a population
distinct from SOX2(+)/NG2(+) OPCs. Together these results reveal
that the ONLR is a mitotically active region of the ON, enriched
with a unique population of SOX2(+)/nestin(+)/GFAP(+) cells,
consistent with other NPC niches in the central nervous system
(CNS).
[0114] CNS-NPC niches have a vascular plexus (Shen et al., 2008;
Tavazoie et al., 2008). The ONLR vasculature is a plexus
(schematic, FIG. 1A) derived from three distinct vascular supplies:
retina, choriocapillaris and optic nerve. Rat ONLR vasculature was
evaluated using two-photon microscopy, and mouse ONLR vasculature
by serial section and reconstruction using neuroleucida (MBF;
Williston VT) following filling with a fluorescein-gelatin mixture
(rat vasculature is shown in FIG. 1C) (Nicholson et al., 2012).
ONLR vascular complexity is greater than that of the distal ON
(FIG. 1C; quantification in 1C inset) and similar to the adjacent
retina (FIG. 1C inset; data adapted from Balaratnasingam et al.,
2014). The ONLR's increased vascular complexity is another
characteristic of CNS-NPC niches (Shen et al., 2008; Tavazoie et
al., 2008).
C. NPCs are Concentrated in the ONLR
[0115] Cells from ONLR (the first 500 um of ON behind the globe)
and distal ON (>2 mm from the globe) tissues were dissociated
from young (PN 20 d) and adult (60 d) animal. Cells were cultured
utilizing medium supplemented with FGF2 and Matrigel. Without
Matrigel, there was poor growth from both laminar and ON cultures
(FIG. 2A; ONLR), with cells on adherent surfaces expressing either
GFAP or O.sub.4 (FIG. 2A, inset) and were morphologically similar
to astrocytes and oligodendrocytes. Optimal propagation of ONLR
cultures required concentrations of 10-15% Matrigel (methods) (FIG.
2B). Distal ON cultures consistently yielded few colonies, even
with Matrigel (FIG. 2E, in red).
[0116] Matrigel-supplemented ONLR cells initially formed flattened
colonies (FIG. 2B), containing numerous cells with a NPC-like
appearance, although cultures were consistent with a mixed cell
population. ONLR colonies on low adhesion medium from the second
passage onward formed neurospheres after dissociation (FIG. 2C),
similar to those seen with NPC derived from the SVZ (FIG. 2C;
inset). Neurospheres regrown on coated plates developed cells with
fine processes (FIG. 2B, inset). Two major cellular populations
were detected: a minor fraction (10% of cells) with NPC-like
morphology (FIG. 2B), and these were shown to be mitotically active
by Ki67 staining (data not shown), as well as nestin(+) and
SOX2(+). These cells co-expressed nestin and SOX2 (FIG. 2D, and
left inset), SOX2 and GFAP (FIG. 2F), and A2B5/SOX2(+) (FIG. 2G;
arrows). The ONLR NPC-like cells provided a continual source of
astrocytes, distinguishable by their morphology and GFAP
immunostaining. Along with astrocytes, NG2(+)/nestin(-) cells were
detected (arrowhead, FIG. 2H). As nestin(+) cells gradually
acquired NG2 expression, their nestin signal progressively
declined. This data is consistent with the in vivo results shown in
FIGS. 1K and L.
[0117] While many nestin(+)/SOX2(+) and SOX2(+)/NG2(+) cells were
seen in culture (FIG. 2I), the loss of nestin in NG2(+) cells,
coupled with the enhanced ability of ONLR cultures to form
neurospheres, suggests that ONLR-nestin(+)/SOX2(+) cells are
distinct from the NG2(+)-OPC population but capable of giving rise
to SOX2(+)/NG2(+)OPCs. This observation is important because it
suggests that ONLR-nestin expressing cells are at a more primitive
progenitor stage than OPCs. ONLR cultures gave rise to rare
(0.016%) NeuN/Tuj1(+) cells (FIG. 2K). Thus, ONLR
Sox2/Nestin/GFAP(+) cells represent a distinct NPC population that
can potentially give rise to all neural cell types, including OPCs.
ONLR cultures supplemented with 10% FBS generated either GFAP(+)
astrocyte-type cells (FIG. 2L, in green) or O.sub.4(+) membranous
oligodendrocyte-type cells (FIG. 2L, in red). ONLR-NPCs are thus
pro-gliogenic, with the propensity to differentiate into macroglia
in vivo, in culture, but in culture can also give rise to
neurons.
[0118] Despite the ONLR's enhanced ability to form neurospheres,
most (90%) ONLR-derived cultured cells were distinctly different
from cells with NPC morphology. These were flattened, often
stellate in appearance (FIG. 2J), A2B5(+)/SOX2(-)/nestin(-), making
them distinct from NG2(+) OPCs (FIG. 2J, in green). Culture results
are summarized in FIG. 2M.
[0119] ONLR cell neurosphere-forming ability was quantified using
2.sup.nd subculture passage-dissociated cells in low adhesion
medium. ONLR cultures generated neurospheres (FIG. 2C)
co-expressing NPC markers nestin and SOX2. 0.016-0.03% of ONLR
cells could generate neurospheres (graph FIG. 2E). NPC frequency in
the ONLR population is similar to that previously reported for
hippocampus and subcortical white matter (WM) (Lojewski et al.,
2014). Dissociated distal ON cells were capable of generating less
than 1 neurosphere per 10.sup.6 cells (0.0007-0.0009%; FIG. 2E, red
bars; n=3 experiments), implying that cells in the distal ON of
both young and old animals have little self-renewal capacity.
ONLR-neurospheres could be generated from both 17 d pups and mature
(60 d) animals, but ONLR from mature animals generated fewer
neurospheres compared with pups (FIG. 2E, compare blue bars in pup
vs adult). A diminished capacity of cells from mature animals to
generate neurospheres in vitro corresponds with the in vivo data
showing reduced nestin expression and declining numbers of SOX2(+)
cells in the ONLR of aged animals (FIGS. 5A and 5B), suggesting
that this progenitor cell population declines during aging.
[0120] NPC-associated gene expression was compared from adult mouse
(60 d) retina, ONLR and ON tissues via rq-PCR (FIG. 2N). ONLR
tissue expressed high nestin, Sox1- and -2 and Msi-1 levels (FIG.
2N). While ONLR expressed higher levels of Pax6, Brn3b and Shh,
than did ON (compare ON and ONLR; FIG. 2N), this expression may be
due to ONLR sample contamination due to the ONLR's retinal
proximity (FIG. 2N; compare retina and ONLR).
D. Transgenic Confirmation of ONLR-NPCs
[0121] A number of the current generation of commercially available
SOX2 antibodies cross-react with other SOX family members
(unpublished data; for comparison see Tiwari et al., 2014). A
cross-reacting SOX2 mouse monoclonal antibody initially resulted in
overestimates of ONLR and ON-SOX2(+) nuclear numbers. Later studies
utilized a rabbit polyclonal antibody that confirmed the relative
SOX2 expression pattern seen in the transgenic experiments
(below).
[0122] In vivo SOX2 expression was confirmed in ONLR and ONs using
a reporter mouse strain expressing Cre under control of the SOX2
promotor, where Cre expression is further restricted by the use of
the ER2 receptor that binds only tamoxifen or its active
metabolite, causing activation only in cells with active SOX2. This
mutant gene is referred to as SOX2-Cre. This enabled selective
identification of the timing of postnatal SOX2 activity, and also
allowed utilization of constructs whose expression would be fatal
during embryogenesis (see below).
[0123] In one set of experiments, SOX2-Cre mice were crossed with a
strain expressing td-Tomato reporter- and incorporating a FLOXed
GFP-protein constructed in the ROSA26 locus (referred to as
SOX2-GFP mice). SOX2-GFP mice constitutively express td-tomato,
expressing GFP only following tamoxifen or 4-OH tamoxifen induction
in SOX2(+) cells and their descendants (FIG. 3A). This enabled
precise identification of the SOX2 expressing cells and their
subsequent progeny post-injection.
[0124] Tamoxifen was administered to 19 d PN animals (schematic,
FIG. 3A). Robust GFP expression was seen in hippocampus 14 d
post-administration (FIG. 3B). SOX2-GFP expression 14 d
post-tamoxifen was localized to the ONLR but was indistinct (FIG.
3C). A 30 d interval following injection was necessary to identify
distinct cell patterns (FIG. 3D). Recombination efficiency in the
lamina 30 d post-induction was .about.40%, based on the number of
GFP(+) cells expressing on the td-Tomato background (Badea et al.,
2003). 30 d post-tamoxifen, ONLR-GFP expression was identifiable in
processes transverse to the long axis of the nerve (3D: ONLR). In
the ONLR, GFP co-localized with nestin (FIG. 3E; arrows), but not
in the anterior ON. ONLR 30 um thick cross section Z-stack
reconstructions revealed individual large GFP(+) cells (FIG.
3F)
[0125] GFP expression was also present in the anterior ON in cells
consistent with NG2 morphology at 30 d (FIG. 3D; arrows). These
cells co-expressed NG2 and GFP (3D inset; arrows). SOX2-driven GFP
expression 30 d post-induction was also present in structures
parallel with the long axis of the ON, consistent with
axon-myelinating segments (FIG. 3D; arrowheads), and co-localizing
with antibody to myelin basic protein (MBP: data not shown). Below
the ONLR, GFAP(+)/GFP(+) cells were identified (FIG. 3G; arrow) as
well as GFP(-)/GFAP(+) cells consistent with astrocytic morphology
(FIG. 3G; arrowheads). The data supports the hypothesis that
ONLR-SOX2/nestin(+) NPCs can give rise postnatally to both
ON-NG2(+) OPCs, as well as directly to astrocytes. These
differentiating daughter cells can migrate into the anterior ON,
ultimately generating mature glial forms. This functional strategy
enables focused ON anterior myelination and astrocyte production
during the late postnatal ON growth phase. Resident SOX2(+)/NG2(+)
cells enables oligodendrocyte replacement in the mature animal, as
previously noted (Kang et al., 2010).
[0126] The effect of postnatal SOX2-NPC ablation in the ONLR was
examined using transgenic animals constructed with the ER2-SOX2-Cre
transgene and the FLOXed diphtheria toxin A (DTA) protein in the
ROSA26 locus (referred to as SOX2-DTA animals). Cre-based
recombination induces DTA protein (Saito et al., 2001), enabling
selective deletion of SOX2-expressing cells following tamoxifen
administration. SOX2-DTA animals thus provide a highly sensitive
tool for analyzing effects of ONLR-SOX2(+) cell loss, since DTA
ablates them. Local administration of 4-hydroxytamoxifen (4OHT) via
retrobulbar injection, enabled selective elimination of
SOX2-expressing cells in one eye (Bernstein et al, manuscript in
preparation). Local administration efficacy was confirmed using
SOX2-GFP animals (FIG. 3D-F).
[0127] ON morphology was compared between 4OHT and contralateral
vehicle (Kolliphor) treated animals 14 d and 30 d
post-administration. Ocular tissues were compared for size and
appearance (FIG. 4A). Post-fixed tissues were evaluated for ONLR
SOX2- (see FIGS. 4F and G) and nestin- (FIGS. 4D and E) expression.
ON ultrastructure was examined using transmission electron
microscopy (TEM) (FIGS. 4I and L).
[0128] Nerves of SOX2-DTA animals given vehicle were normal
appearing (see FIGS. 4B and C: `V`), with a normal contralateral
optic tract (FIGS. 4B and C: arrow). In contrast, 4OHT-treated ONs
14 d post-induction were hypoplastic (FIGS. 4B and C: compare V and
4OHT: arrowheads indicate nerve of the 4OHT-treated side). The
optic tracts associated with 4OHT-treated nerves also had a reduced
appearance, compared with vehicle-treated ON tracts (FIGS. 4B and
C: arrow).
[0129] DTA-induced NPC cell loss post-4OHT was assessed in 33 d PN
animals. There was selective loss of nestin and SOX2 signal in 4OHT
treated ONLR- and ONs (FIGS. 4E; nestin and G; SOX2), compared with
vehicle-treated nerves (FIGS. 4D; nestin and F; SOX2). Thus,
deletion of SOX2(+) ONLR cells drastically reduced anterior nestin
expression and altered normal ONLR structure.
[0130] Quantification of SOX2 nuclear expression at 4 d post-4OHT
administration revealed a loss of over 2/3 of SOX2 nuclei in the
ONLR compartment, compared with WT animals (FIG. 4G inset:
6.9.+-.1.9% SOX2(+) nuclei in the ONLR of 4-OHT treated SOX2-DTA
animals v. 21.8.+-.4.9% in the ONLR of WT; n=3 animals group,
.+-.sem). These data suggest that the loss of SOX2(+) nuclei
post-induction is not due simply to tamoxifen-related drug toxicity
or retrobulbar injection, but rather a loss of NPCs.
[0131] The ONLR extends .about.300 um from the 60 d PN mouse eye
(Wang et al., 2017 and FIG. 1). It was hypothesized that the
postnatal retinal ganglion cell (RGC) axon grows unidirectionally
from the unmyelinated region, with postnatal myelination supplied
by glia derived from ONLR-NPCs. With this pattern, anterior
myelination in the growing ON would occur focally as a distinct
zone directly below the ONLR, as unmyelinated axons emerge from the
lamina. To examine this, myelination patterns were compared in the
anterior- and mid-ON from postnatal SOX2-DTA animals, in which
active ON growth was still occurring (FIG. 4H-K). Animals were
treated at 19 d PN, and evaluated at 33 d PN (14 d post-treatment).
ONs were ultrastructurally analyzed using TEM at their anterior-
(1.5-2 mm from eye) and distal (5 mm from the eye) segments
(schematic, FIG. 4H). WT animals injected either with vehicle or
4OHT showed no gross alterations or significant axonal loss (data
not shown).
[0132] Ultrastructural results in nerves of SOX2-DTA animals
treated with vehicle were similar to those of WT animals (FIGS. 4L,
anterior, and 4K. distal segments). Relative myelin thickness to
axon diameter (G-ratio) was unchanged, and similar at both anterior
and distal segments (FIG. 4N, white bars). In contrast,
4OHT-treated nerves of SOX2-DTA animals revealed axonal
hypomyelination in the anterior ON segment, with a corresponding
increase in the G-ratio (FIG. 4N, black bars: compare anterior ON
(lamina) and distal (optic nerve), along with axonal loss and
reduced packing density (FIG. 4I), compared to vehicle treated
anterior nerve from the same animal (compare FIG. 4L, vehicle, with
4J, 4-OHT treated). Comparing mean relative axon size in the
anterior and distal segments, a trend toward a larger mean axon
diameter was seen in anterior ON segments (FIG. 4M; 522.+-.190 nm
for vehicle v. 771.+-.382 nm for 4OHT), due to a loss of the
smallest axon class (<400 nm diameter) in 4OHT treated nerves
(absence of a black bar in the <400 um size class in the
individual G-ratio axon size graph in FIG. 4O). This suggests that
either small axons are selectively lost with 4OHT treatment, or
there is an increase in the relative diameter of the smallest
axons, possibly due to swelling from intra-axonal fluid
accumulation. The latter explanation is most likely since a
comparison of relative mean axon diameter from the distal ON
segments of both treatment groups revealed little, if any
differences in mean axonal diameter (607.+-.261 nm for vehicle v.
612.+-.228 nm for 4OHT) and a similar packing density. In distal ON
segments of 4-OHT-treated nerves, (FIG. 4J), myelination was
similar to that of vehicle-treated nerves (compare 4J with 4K;
vehicle).
[0133] The G-ratio was calculated for each axon size class in
Vehicle and 4-OHT treated nerves (FIG. 4O). There was an increase
in the G-ratio in every axon size class in 4-OHT treated nerves, an
exception being the loss of the smallest diameter axons (<400 nm
in diameter) in 4-OHT treated ON. 4-OHT treatment resulted in a
mean increase in the G-ratio, indicating a relative thinning of the
myelin sheaths of axons in the anterior region (FIG. 4N; Anterior
ON: 4OHT=0.79.+-.0.04 (sd) v. Vehicle=0.67.+-.0.03 (sd)), compared
with vehicle treated nerves from the same animal (two tailed
t-test; p<0.001). This was not apparent in the distal segment,
where the mean distal ON G-ratio for 4OHT- and vehicle-treated
nerves were similar (FIG. 4N: ON. 4OHT; 0.69.+-.0.08 v. vehicle;
0.66.+-.0.09) from the same animal(s).
[0134] The anterior ON of 4OHT-treated nerves also had decreased
axon packing density (increased distance between axons) and
increased vacuoles were observed in the anterior portion of 4OHT
treated--compared with vehicle-treated nerves from the same animal
(compare axonal packing in FIG. 4I; 4OHT treated, with 4L; vehicle
treated).
[0135] Despite anterior ON hypomyelination, distal axon segment
myelination was similar in both 4OHT- and vehicle-treated nerves
(compare FIGS. 4J, 4OHT-treated and 4L, vehicle-treated,
respectively).
[0136] Randomized axonal counts from six fields revealed a mean
40.5% loss in 4OHT treated ONs, (mean 126.+-.27.6 axons/field in
vehicle v. 76.+-.9.9 axons in 4OHT per 165 um.sup.2 field) from
SOX2-DTA animals (white bars), compared with vehicle treated eyes
(p<0.05, Fisher exact test). These results reveal that selective
postnatal deletion of SOX2(+)/nestin(+) ONLR-NPCs during active ON
growth results in a pattern of regional loss of anterior
myelination. These results are consistent with the hypothesis that,
in addition to axonal loss, ONLR-NPCs contribute to progressive
development and migration of newly formed oligodendrocytes and
astrocytes in the growing postnatal ON.
E. Age-Related Quantitative Changes in NPCs in Rodent and Human
ONLR.
[0137] Adult NPC numbers decline during aging (Encinas et al.,
2011), and ONLR-NPC decline may be associated with the aged ON's
reduced reparative ability (Malik et al., 2014). Age-related
ONLR-NPC-associated changes were quantified in mice by two
approaches: 1) immunohistochemical analysis and quantification of
SOX2 nuclei in the ONLR of 1, 7 and 24 m/o mice (FIG. 5A-C); 2)
nestin mRNA quantification in 1, 6 and 12 m/o mice using rq-PCR
(n=6 animals/prep) (FIG. 5D).
[0138] For 1), SOX2(+) nuclear numbers in ONLR sections were
compared using equivalent relative surface area per region (45,000
um.sup.2.times.3 sections/animal; n=3 animals FIG. 5C).
ONLR-SOX2(+) nuclear numbers declined 73% in 24 m/o animals,
compared with 1 m/o animals (FIG. 5C, black bars: 53.+-.36 v.
197.+-.10; SEM; n=6 nerves/group). Interestingly, ONLR has a
greater number of SOX2(+) nuclei than distal ON, but the number of
SOX2(+) cells in distal ON remains relatively constant during aging
(FIG. 5C; white bars). Similarly, for 2), mouse ONLR nestin mRNA
levels declined by 56% by 12 m/o, compared with nestin mRNA levels
seen in 1 m/o animals (FIG. 5D; compare 1 m/o and 12 m/o). ONLR
thus exhibits an age-dependent decline in both NPC-associated
proteins, suggesting that ONLR-NPCs diminish with age.
F. The Human ONLR Contains an Age-Depletable NPC Niche
[0139] The primate laminar structure is more elaborate than rodents
(Anderson and Hoyt, 1969), but the pattern of overall ONLR
organization is similar in both phylogenetic families (Albrecht,
2008). Young human ONLRs strongly express filamentary nestin and
possess abundant SOX2(+) nuclei, (FIGS. 5A, 7 m/o fetal ONLR and
5B, 9 y/o donors). Younger donors generally showed increased
SOX2(+) nuclear expression compared with tissue from older donors.
However, young donor tissues obtained with prolonged times (>24
hours) from demise until preservation showed reduced SOX2 signal
(compare 5F, 9 y/o and G, 40 y/o). This preservation time-dependent
decline of SOX2 expression resulted in variability between donors
of similar ages.
[0140] Nestin expression was found to be less dependent on time to
preservation, possibly because structural proteins are more stable
than nuclear factors. Densitometric quantification of
age-associated changes in donor human ONLR nestin expression is
shown in FIG. 5E-H. ONLR-nestin expression declined precipitously
with donor age (compare signals from youngest donors: 7 mo fetal in
5E and 9 y/o in 5F, with reduced signal from tissues of
intermediate age donors 40 y/o (FIG. 5G) and 50 y/o (FIG. 5H)).
Filamentous nestin was absent in the ONLR of the oldest donors (5H
inset). Human ONLR-nestin expression declined >90% by age 60 y
(linear regression statistic, r=0.92; FIG. 5I). Tissue from elderly
donors, even those having minimal time from collection to
preservation contained few, if any, ONLR-SOX2(+) nuclei or nestin.
These data suggest that the human ONLR, like mice, contains an
age-depletable cell niche of ONLR-NPCs.
[0141] To confirm the presence of human ONLR-NPCs, ONLRs were
dissociated from freshly obtained human surgical specimens
(approved by the UMB-institutional review board (TRB)). Human
cultures utilized media conditions identical to those used for
mouse ONLR cultivation. Initial growth on adherent plates with
Matrigel-supplemented medium generated mixed cultures, similar to
those seen in rodent. Cells with a bipolar morphology were a
prominent feature in early passage cultures grown on adherent
plates. Neurospheres were generated when cells were regrown in low
adherence conditions (FIG. 5J). Immunostaining of passage 3 human
ONLR-neurospheres yielded cells co-expressing SOX2, GFAP and
nestin, consistent with NPC characteristics (FIGS. 5K and 5L).
G. ONLR Stem Cell Niche Gene Expression
[0142] The ONLR contains a neural stem cell niche. To determine
whether the ONLR stem cell niche plays a role in the prevention or
resistance to glaucoma, gene expression of cells from the niche was
examined. Fresh (<1 hr old) tissue from young (4-6 year old)
male and female rhesus macaque monkeys that previously had no
ocular disease or conditions that would alter ocular gene
expression was isolated. This included the eye and attached optic
nerve. This tissue was kept on ice until further dissection. The
retina, the optic nerve lamina containing the neural stem cell
niche (first 0.5 cm of the optic nerve, containing the optic nerve
head), and distal optic nerve tissues (>1 cm behind the laminar
region) was dissected. These tissues were isolated from the
original tissue and stored at -80.degree. C. until use.
[0143] Total RNA was isolated using a sequential purification via
RNA Bee (guanidinium thiocyanate-phenol), followed by resuspension
in solution provided in the Qiaprep RNA isolation kit. The
resuspended RNA was re-isolated through Qiaprep RNA isolation
columns, yielding ultrapure total RNA. Total RNA was used for
sequence analysis. Two independent analyses were performed. First,
RNA was utilized from one sample of lamina and one sample of optic
nerve. These RNA samples were sent to the Yale sequencing facility
for further processing into complementary strand RNA and illumina
RNA sequencing and sequence comparison using standard sequence
analysis procedures. Total RNA prepared from a new lamina tissue
(the second animal), as well as total RNA from retina from two
animals and two optic nerves were sent to the University of
Maryland, Baltimore, Biocore for further processing into
complementary strand DNA and illumina RNA sequencing. Thus, there
were a total of two retina samples, two lamina samples and two
optic nerve samples used for sequencing. Analysis was performed
using the Qiagen sequence ingenuity program analysis (IPA) analysis
software package. Gene expression was compared by combining CPM
sequence results from retina and optic nerve, and related to lamina
CPM, using the following formula:
Lamina .times. .times. CPM .times. .times. results Retina .times.
.times. 1 .times. .times. CPM + Retina .times. .times. 2 .times.
.times. CPM + Optic .times. .times. Nerve = Laminar .times. .times.
niche .times. / .times. surrounding .times. .times. tissue .times.
.times. ratio ##EQU00001##
[0144] Results from genes associated with growth factors were
identified and then grouped together. The growth factors with
Lamina/Retina+Optic Nerve ratios >3.0 were further identified.
Two groups of genes were thus established: 1) total gene
comparison, and 2) growth-factor associated genes that are
differentially expressed at higher levels in the lamina stem cell
niche.
[0145] Genes with laminar niche/surrounding tissue ratios
.gtoreq.3.0 and Laminar CPM counts .gtoreq.90 were identified. The
chromosomal localization of each gene was confirmed using Genecards
(see the website having the URL ending with genecards.org/), and a
Google search was performed to determine whether this chromosomal
location was reported to be associated with the optic nerve disease
glaucoma (either primary open-angle glaucoma, juvenile open-angle
glaucoma, normal tension glaucoma, or congenital glaucoma). The
results presented in Table 7 are strong evidence that the ONLR
neural stem cell niche expresses genes that are responsible for
resistance to developing glaucoma, and also identify the growth
factors and related proteins that are involved in this resistance.
The products of these genes can be either prepared as the active
peptide, or the whole protein, and administered as a cocktail of
the individual factors, such as in the artificial ONLR-NPC extracts
of the invention defined herein.
TABLE-US-00007 TABLE 7 ONLR stem cell niche gene expression Ratio =
name 233_ret 240_ret 241_lam 242_on gene Lam/Ret + ON Name
ENSMMUG00000005603 0 0 90 1 UCMA 90 Upper Zone of Growth Plate and
Cartilage Matrix Associated Protein ENSMMUG00000005068 1 0 94 1
GFI1 62.66667 Growth Factor Independent 1 Transcriptional Repressor
ENSMMUG00000002875 5 3 194 2 FGF14 32.33333 Fibroblast Growth
Factor 14 ENSMMUG00000004919 0 0 95 3 FGFR4 31.66667 Fibroblast
Growth Factor Receptor 4 ENSMMUG00000000896 6 13 534 10 TGFB1I1
27.38462 Transforming growth factor beta-1-induced transcript 1
protein ENSMMUG00000005353 2 0 92 3 PDGFD 23 Platelet Derived
Growth Factor D/Spinal Cord-Derived Growth Factor B
ENSMMUG00000002909 0 3 148 5 PGF 22.76923 Placental Growth Factor
ENSMMUG00000003468 5 3 139 5 GADD45B 15.44444 Growth arrest and DNA
damage-inducible protein GADD45 beta ENSMMUG00000000332 10 33 596
18 TGFBI 15.08861 Transforming growth factor beta-1 proprotein:
Both latency and factors ENSMMUG00000001153 0 1 292 21 PDGFRL
13.5814 Platelet-derived growth factor receptor-like protein
ENSMMUG00000002235 25 24 219 0 IGF1 8.938776 Insulin growth
factor-1 ENSMMUG00000003748 2 5 137 14 VEGFC 7.828571 Vascular
Endothelial Growth Factor C ENSMMUG00000000401 59 81 594 28 GAS8
6.061224 Dynein regulatory complex subunit 4 ENSMMUG00000001336 13
3 235 40 GHR 4.895833 Growth Hormone Receptor ENSMMUG00000004594 22
25 128 11 CGRRF1 3.710145 Cell Growth Regulator With Ring Finger
Domain 1 ENSMMUG00000000949 61 41 336 56 FGF12 3.140187 Fibroblast
growth factor 12 name Chromosome Disease linkage ENSMMUG00000005603
10p13 Normal Tension glaucoma ENSMMUG00000005068 1p22.2 Primary
open-angle glaucoma (POAG) ENSMMUG00000002875 13q33.1
Pseudoexfoliative glaucoma ENSMMUG00000004919 5q35.2 POAG
ENSMMUG00000000896 16p11.2 Congenital glaucoma ENSMMUG00000005353
11q22.3 POAG ENSMMUG00000002909 14q24.3 Primary congenital glaucoma
(GLC3C) ENSMMUG00000003468 19p13.3 GLC10 glaucoma gene site
ENSMMUG00000000332 19q13.2 POAG ENSMMUG00000001153 8p22 POAG
ENSMMUG00000002235 12p23.2 increased levels enhance retinal
ganglion cell survival/reduce glaucoma loss (Ma et al., 2015)
ENSMMUG00000003748 4q34.3 POAG ENSMMUG00000000401 2p23.3 POAG
ENSMMUG00000001336 5p13.1 POAG ENSMMUG00000004594 14q22.2 glaucoma
locus GLC3C ENSMMUG00000000949 3q28-q29 Low tension glaucoma
[0146] This data demonstrates that the rodent and human postnatal
ONLR contains nestin(+)/SOX2(+)/NG2(-) NPCs that give rise to
oligodendrocyte progenitor cells (OPCs) and astrocytes.
Self-replenishing neural-derived cells that are found within a
dedicated vascular plexus give rise to culturable neurospheres,
express multiple stem cell marker proteins, differentiate into
multiple neural forms, and are age-depleted; these cells that
fulfill the criteria of adult NPCs. ONLR-NPCs are distinct from the
nestin(-)/NG2(+) ON-OPCs, which are distinguished by their relative
inability to generate neurospheres, grow poorly, and give rise
mainly to oligodendrocytes. ONLR-NPCs resemble `atypical
astrocytes` previously reported to occur in the ONLR region (Wang
et al., 2017). It is hypothesized that ONLR-NPCs likely perform
multiple functions: during the early postnatal period, these cells
enable focal myelination in the growing axons, enhancing
directional polarization of axonal myelination and also generate
astrocytes for the growing ON. ONLR-NPCs may provide a gliogenic
processing center during ON growth, contributing to myelination and
astrocyte production. In the mature ON, ONLR-NPCs enhance cellular
replacement from stress-related loss and may also supply specific
growth factors needed for normal axonal survival and function,
similar to that seen in other CNS niches (Lu et al., 2003).
[0147] NG2(+) cells comprise 2-8% of the ON (Kang et al., 2010),
yet the ON has reduced capacity for remyelination compared with
spinal cord (SC) (Lachapelle et al., 2005). If ON remyelination
capacity arises from the ONLR-NPC population, then effective repair
can occur only in the anterior nerve, while SC-NPC remyelination
can occur throughout the spinal cord.
[0148] The increased ratio of SOX2(+) nuclei to ONLR total nuclei
in 2 m/o adult mice, vs the distal ON, is maintained through at
least 7 PN months. Supporting these findings, the mouse ONLR formed
neurospheres at a much higher frequency compared with distal ON
tissue (graph, FIG. 2E). The ONLRs ability to form neurospheres was
present in both young and adult animals. It is also present in ONLR
from young human donors. The culture data confirms that that
SOX2(+) cells in human and rodent ONLR-derived neurospheres
co-express nestin(+). These data support the hypothesis that the
SOX2(+)/nestin(+) cells present in the mammalian ONLR likely
represent a replicating NPC population, endowing the ONLR with a
greater response repertoire than that of the distal ON.
[0149] Many NG2(+) cells were generated during ONLR-NPC culture,
suggesting a preference for NPC>OPC differentiation. Terminal
differentiation of cells from ONLR cultures generated both
astrocytes and oligodendrocytes, with few (<3%) NeuN(+) or
Tuj1(+) cells. This is consistent with the ONLR-NPCs' hypothesized
role in glial replenishment. This was confirmed by ON
immunohistochemistry following induction of SOX2-GFP double
mutants, which yields GFP(+)/NG2(-) cells in the ONLR, and GFP(+)
cells with both astrocytic and oligodendroglial cell morphology in
the anterior ON, whereas GFP(+) cells in the distal portion of same
nerves exhibit only either NG2(+) or oligodendrocyte morphology.
These results support the hypothesis that one role for ONLR-NPCs is
to enable in vivo generation of mature glia.
[0150] Postnatal axonal growth can occur at specific stress points
located at myelin: unmyelinated junctions (Goldberg, 2003). Since
the region adjacent to the ONLR is where RGC axonal myelination
begins, these results suggest that postnatal ON growth occurs by
axonal extension from the unmyelinated retina, with myelination
directly associated with emergence from the ONLR. ONLR-NPCs provide
a source of oligodendrocytes and astrocytes during intensive
postnatal ON growth. Without adequate ONLR-NPCs, axons either
remain hypomyelinated or die. The data herein show both axonal loss
and anterior hypomyelination following selective depletion of
ONLR-NPCs following exposure to 4-OHT in SOX2-DTA reporter mice. In
contrast, more distal axonal segments had a nearly normal
myelination pattern.
[0151] In adults, overall oligodendrocyte generation declines, but
the ONLR continues to have an increased Ki67(+) mitotic index,
suggesting that the ONLR niche continues to contribute to adult
glial cell replacement. It is hypothesized that ONLR-NPCs have
distinct roles during early postnatal and adult stages: in the
young, ONLR-NPCs contribute to ON growth, while in the adult they
contribute to glial cell replacement.
[0152] Mouse ONLR-SOX2(+) nuclear numbers decline during aging, but
remain relatively constant in the distal ON. An age-related decline
in human ONLR-nestin expression is demonstrable (see FIG. 5i).
These data indicate age-dependent ONLR-NPC loss. Human ONLR-NPCs
are ultimately lost during aging, and this loss may alter the
balance between degeneration, repair and the emergence of
age-related ON diseases.
[0153] An important age-associated ON disease is open-angle
glaucoma (OAG) (Lee et al., 2014), a progressive neuropathy
associated with the ONLR. Progressive resistance to glaucoma
treatment occurs in elderly individuals (Rossetti et al., 2010).
ONLR-NPC depletion may contribute to OAG progression by reducing
gliogenic support and associated neurotrophic factors such as
brain-derived growth factor (BDNF), important for retinal ganglion
cell (RGC) survival under stress, and which is secreted by cultured
lamina-derived cells (Lambert et al., 2004). It is hypothesized
that ONLR-NPCs present in younger individuals may protect against
disease progression, and the age-related loss of ONLR-NPCs may
contribute to disease progression via the inability to repair
stress-related damage.
[0154] In summary, the ONLR in both humans and mice is not a
non-proliferative barrier, but rather contains an NPC niche, which
may have a role in both postnatal ON development and in adult ON
support and repair. Age-related ONLR-NPC loss likely contributes to
ON diseases such as OAG and understanding the roles of ONLR-NPCs
enables new therapeutic strategies to treat ON disease.
[0155] While the invention has been described with reference to
certain particular embodiments thereof, those skilled in the art
will appreciate that various modifications may be made without
departing from the spirit and scope of the invention. The scope of
the appended claims is not to be limited to the specific
embodiments described.
REFERENCES
[0156] All patents and publications mentioned in this specification
are indicative of the level of skill of those skilled in the art to
which the invention pertains. Each cited patent and publication is
incorporated herein by reference in its entirety. All of the
following references have been cited in this application: [0157]
Ahlenius, H., Visan, V., Kokaia, M., Lindvall, O., and Kokaia, Z.
(2009). Neural stem and progenitor cells retain their potential for
proliferation and differentiation into functional neurons despite
lower number in aged brain. J Neurosci 29, 4408-4419. [0158]
Albrecht, M. C. (2008). Comparative anatomy of the optic nerve head
and inner retina in non-primate animal models used for glaucoma
research [In Process Citation]. Open Ophthalmol J 2, 94-101. [0159]
Almasieh, A., Wilson, A. S., Morquette, B., Cuega Vargas, J. L.,
and DiPolo, A. (2012). The molecular basis of retinal ganglion cell
death in glaucoma. Progress in retinal and eye research 31,
152-181. [0160] Anderson, D. R., and Hoyt, W. F. (1969).
Ultrastructure of intraorbital portion of human and monkey optic
nerve. Arch Ophthalmol 82, 506-530. [0161] Badea, T. C., Wang, Y.,
and Nathans, J. (2003). A noninvasive genetic/pharmacologic
strategy for visualizing cell morphology and clonal relationships
in the mouse. J Neurosci 23, 2314-2322. [0162] Balaratnasingam, C.,
Kang, M. H., Yu, P., Chan, G., Morgan, W. H., Cringle, S. J., and
Yu, D. Y. (2014). Comparative quantitative study of astrocytes and
capillary distribution in optic nerve laminar regions. Exp Eye Res
121, 11-22. [0163] Cavazzin, C., Ferrari, D., Facchetti, F.,
Russignan, A., Vescovi, A. L., La Porta, C. A., and Gritti, A.
(2006). Unique expression and localization of aquaporin-4 and
aquaporin-9 in murine and human neural stem cells and in their
glial progeny. Glia 53, 167-181. [0164] Encinas, J. M., Michurina,
T. V., Peunova, N., Park, J. H., Tordo, J., Peterson, D. A.,
Fishell, G., Koulakov, A., and Enikolopov, G. (2011).
Division-coupled astrocytic differentiation and age-related
depletion of neural stem cells in the adult hippocampus. Cell Stem
Cell 8, 566-579. [0165] Goldberg, J. L. (2003). How does an axon
grow? Genes & Development 17, 941-958. [0166] Gorris, R.,
Fischer, J., Erwes, K. L., Kesavan, J., Peterson, D. A., Alexander,
M., Nothen, M. M., Peitz, M., Quandel, T., Karus, M., et al.
(2015). Pluripotent stem cell-derived radial glia-like cells as
stable intermediate for efficient generation of human
oligodendrocytes. Glia 63, 2152-2167. [0167] Kang, S. H., Fukaya,
M., Yang, J. K., Rothstein, J. D., and Bergles, D. E. (2010). NG2+
CNS Glial Progenitors Remain Committed to the Oligodendrocyte
Lineage in Postnatal Life and following Neurodegeneration. Neuron
68, 668-681. [0168] Lachapelle, F., Bachelin, C., Moissonnier, P.,
Nait-Oumesmar, B., Hidalgo, A., Fontaine, D., and Baron-Van
Evercooren, A. (2005). Failure of remyelination in the nonhuman
primate optic nerve. Brain Pathol 15, 198-207. [0169] Lambert, W.
S., Clark, A. F., and Wordinger, R. J. (2004). Neurotrophin and Trk
expression by cells of the human lamina cribrosa following
oxygen-glucose deprivation. BMC Neurosci 5, 51-51. [0170] Lee, J.
M., Caprioli, J., Nouri-Mahdavi, K., Afifi, A. A., Morales, E.,
Ramanathan, M., Yu, F., and Coleman, A. L. (2014). Baseline
prognostic factors predict rapid visual field deterioration in
glaucoma. Invest Ophthalmol Vis Sci 55, 2228-2236. [0171] Levin, L.
A. (2005). Pathophysiology of the progressive optic neuropathy of
glaucoma. Ophthalmol Clin North Am 18, 355-335v. [0172] Lojewski,
X., Hermann, A., Wegner, F., Arauzo-Bravo, M. J., Hallmeyer-Elgner,
S., Kirsch, M., Schwarz, J., Scholer, H. R., and Storch, A. (2014).
Human adult white matter progenitor cells are multipotent
neuroprogenitors similar to adult hippocampal progenitors. Stem
Cells Transl Med 3, 458-469. [0173] Lu, P., Jones, L. L., Snyder,
E. Y., and Tuszynski, M. H. (2003). Neural stem cells
constitutively secrete neurotrophic factors and promote extensive
host axonal growth after spinal cord injury. Exp Neurol 181,
115-129. [0174] Ma J, Guo C, Guo C, Sun Y, Liao T, Beattie U, et
al. (2015) Transplantation of Human Neural Progenitor Cells
Expressing IGF-1 Enhances Retinal Ganglion Cell Survival. PLoS ONE
10(4): e0125695. https://doi.org/10.1371/journal.pone.0125695.
[0175] Malik, M. T., Healy, B. C., Benson, L. A., Kivisakk, P.,
Musallam, A., Weiner, H. L., and Chitnis, T. (2014). Factors
associated with recovery from acute optic neuritis in patients with
multiple sclerosis. Neurology 82, 2173-2179. [0176] Nicholson, J.
D., Puche, A. C., Guo, Y., Weinreich, D., Slater, B. J., and
Bernstein, S. L. (2012). PGJ2 Provides Prolonged CNS Stroke
Protection by Reducing White Matter Edema. PLoS One 7, e50021.
[0177] Park, D., Xiang, A. P., Mao, F. F., Zhang, L., Di, C. G.,
Liu, X. M., Shao, Y., Ma, B. F., Lee, J. H., Ha, K. S., et al.
(2010). Nestin Is Required for the Proper Self-Renewal of Neural
Stem Cells. Stem Cells 28, 2162-2171. [0178] Rossetti, L., Goni,
F., Denis, P., Bengtsson, B., Martinez, A., and Heijl, A. (2010).
Focusing on glaucoma progression and the clinical importance of
progression rate measurement: a review. Eye (Lond) 24 Suppl 1,
S1-S7. [0179] Saito, M., Iwawaki, T., Taya, C., Yonekawa, H., Noda,
M., Inui, Y., Mekada, E., Kimata, Y., Tsuru, A., and Kohno, K.
(2001). Diphtheria toxin receptor-mediated conditional and targeted
cell ablation in transgenic mice. Nat Biotech 19, 746-750. [0180]
Shen, Q., Wang, Y., Kokovay, E., Lin, G., Chuang, S. M., Goderie,
S. K., Roysam, B., and Temple, S. (2008). Adult SVZ stem cells lie
in a vascular niche: a quantitative analysis of niche cell-cell
interactions. Cell Stem Cell 2008 Sep. 11; 3, 289-300. [0181]
Stoll, E. A., Habibi, B. A., Mikheev, A. M., Lasiene, J., Massey,
S. C., Swanson, K. R., Rostomily, R. C., and Horner, P. J. (2011).
Increased re-entry into cell cycle mitigates age-related neurogenic
decline in the murine subventricular zone. Stem Cells 29,
2005-2017. [0182] Tavazoie, M., Van der Veken, L., Silva-Vargas,
V., Louissaint, M., Colonna, L., Zaidi, B., Garcia-Verdugo, J. M.,
and Doetsch, F. (2008). A specialized vascular niche for adult
neural stem cells. Cell Stem Cell 3, 279-288. [0183] Tiwari, S.,
Dharmarajan, S., Shivanna, M., Otteson, D. C., and Belecky-Adams,
T. L. (2014). Histone deacetylase expression patterns in developing
murine optic nerve [In Process Citation]. BMC Dev Biol 14, 1-18.
[0184] Wang, R., Seifert, P., and Jakobs, T. C. (2017). Astrocytes
in the Optic Nerve Head of Glaucomatous Mice Display a
Characteristic Reactive Phenotype Astrocytes in the Optic Nerve
Head of Glaucomatous Mice. Investigative Ophthalmology & Visual
Science 58, 924-932.
Sequence CWU 1
1
31120DNAArtificial Sequencechemically synthesized primer sequence
1gaggcgctgg aacagagatt 20221DNAArtificial Sequencechemically
synthesized primer sequence 2cacagccagc tggaactttt c
21321DNAArtificial Sequencechemically synthesized primer sequence
3ggcagtcata caaaagttgg c 21423DNAArtificial Sequencechemically
synthesized primer sequence 4gtacagtatt tatcgtccgc aga
23521DNAArtificial Sequencechemically synthesized primer sequence
5caggagttgt caaggcagag a 21620DNAArtificial Sequencechemically
synthesized primer sequence 6cttaagcctc gggctccaaa
20720DNAArtificial Sequencechemically synthesized primer sequence
7gaccaacccc cgbggcgcac 20820DNAArtificial Sequencechemically
synthesized primer sequence 8tgggcccgaa tcattgtctg
20920DNAArtificial Sequencechemically synthesized primer sequence
9gccttgttga tatcccgagt 201021DNAArtificial Sequencechemically
synthesized primer sequence 10gcccatcatg atcatctctc g
211120DNAArtificial Sequencechemically synthesized primer sequence
11tgtgtaagca gagctcatgc 201218DNAArtificial Sequencechemically
synthesized primer sequence 12catagggtct cggggtct
181320DNAArtificial Sequencechemically synthesized primer sequence
13tgagagggat agctgtgagc 201419DNAArtificial Sequencechemically
synthesized primer sequence 14gttccaagcc acgacctac
191520DNAArtificial Sequencechemically synthesized primer sequence
15cacgtacagt gctttgccac 201620DNAArtificial Sequencechemically
synthesized primer sequence 16aactccgccc attcactgac
201721DNAArtificial Sequencechemically synthesized primer sequence
17cgtaagtcct tcaccagctt g 211821DNAArtificial Sequencechemically
synthesized primer sequence 18gaatccaaag ctcacatcca c
211920DNAArtificial Sequencechemically synthesized primer sequence
19gagacttcac caggggagat 202021DNAArtificial Sequencechemically
synthesized primer sequence 20aaaaccacat gcttgccatc c
212121DNAArtificial Sequencechemically synthesized primer sequence
21cagcaaggac actgagcaag a 212221DNAArtificial Sequencechemically
synthesized primer sequence 22gcccctcctg ttattatggg g
212322DNAArtificial Sequencechemically synthesized primer sequence
23gcggtctggc agtaaaacta tc 222423DNAArtificial Sequencechemically
synthesized primer sequence 24gtgaaacagc attgctgtca ctt
232517DNAArtificial Sequencechemically synthesized primer sequence
25cgacctgcag gtcctcg 172624DNAArtificial Sequencechemically
synthesized primer sequence 26ctcgagtttg tccaattatg tcac
242724DNAArtificial Sequencechemically synthesized primer sequence
27ccaaagtcgc tctgagttgt tatc 242820DNAArtificial Sequencechemically
synthesized primer sequence 28gagcgggaga aatggatatg
202921DNAArtificial Sequencechemically synthesized primer sequence
29ctctgctgcc tcctggcttc t 213019DNAArtificial Sequencechemically
synthesized primer sequence 30tcaatgggcg ggggtcgtt
193121DNAArtificial Sequencechemically synthesized primer sequence
31cgaggcggat cacaagcaat a 21
* * * * *
References